WO2022089602A1 - New target for diagnosis and treatment of chemotherapy drug-resistant small cell lung cancer and application thereof - Google Patents

New target for diagnosis and treatment of chemotherapy drug-resistant small cell lung cancer and application thereof Download PDF

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WO2022089602A1
WO2022089602A1 PCT/CN2021/127538 CN2021127538W WO2022089602A1 WO 2022089602 A1 WO2022089602 A1 WO 2022089602A1 CN 2021127538 W CN2021127538 W CN 2021127538W WO 2022089602 A1 WO2022089602 A1 WO 2022089602A1
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ggps1
rab7a
lung cancer
small cell
cell lung
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French (fr)
Chinese (zh)
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季红斌
郭晨晨
胡良
万睿婕
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中国科学院分子细胞科学卓越创新中心
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Definitions

  • the discovery belongs to the fields of molecular biology and oncology, and more particularly, the present invention relates to a new target for diagnosis and treatment of chemotherapy-resistant small cell lung cancer and its application.
  • Lung cancer is a malignant tumor with high morbidity and mortality.
  • lung cancer is the leading cancer causing death in the world today.
  • many countries have reported that the incidence and mortality of lung cancer have increased significantly.
  • China due to a large number of smokers and the continuous deterioration of the environment, lung cancer has become the highest incidence of cancer in China.
  • Many factors hinder the improvement and perfection of lung cancer treatment programs, and the current five-year survival rate is still less than 10%.
  • Non-small cell lung cancer can be subdivided into lung adenocarcinoma, lung squamous cell carcinoma and large cell lung cancer.
  • Small cell lung cancer is the subtype of lung cancer with the highest degree of malignancy and the worst prognosis due to its high metastasis and the limitation of single treatment. Small cell lung cancer mostly occurs in the central part of the lung, grows rapidly and metastasizes earlier.
  • Etoposide (VP-16) combined with cisplatin (DDP) has become the standard first-line treatment because of its good chemotherapy effect and less toxicity.
  • Chemotherapy can relieve the symptoms and prolong the survival period of patients with small cell lung cancer, but drug resistance occurs quickly, so the current survival rate of small cell lung cancer patients is not ideal, and the 5-year survival rate is less than 5%.
  • the lack of access to tumor samples from patients with small cell lung cancer has led to a serious lag in understanding its pathogenesis. The survival and treatment methods of patients with small cell lung cancer have not changed much.
  • Tumor cells have complex and diverse metabolic pathways to support their own growth and proliferation. Tumor cells in particular rely on diverse metabolic pathways to synthesize substances and energy required for their rapid proliferation. Given that tumor cells are overly reliant on reprogrammed metabolic pathways to support their survival and proliferation, targeted therapy can target the metabolic specificity of tumor cells. Therefore, it is particularly important to study the relationship between tumor metabolism and its survival and proliferation.
  • the purpose of the present invention is to provide a new target for diagnosing and treating chemotherapy-resistant small cell lung cancer and its application.
  • the present invention relates to the discovery and application of a new therapeutic target for chemotherapy-resistant small cell lung cancer.
  • Disclosed is a method for constructing a chemoresistance mouse model of small cell lung cancer; it is also disclosed that small molecule inhibitor statins selectively inhibit chemoresistance through geranylgeranyl pyrophosphate (GGPP)-RAB7A-autophagy pathway Growth of small cell carcinoma; also disclosed that knocking down the gene expression of GGPP synthase (GGPS1) or RAB7A can selectively inhibit the proliferation of chemotherapy-resistant small cell carcinoma tumors; also disclosed that statins and their combination with chemotherapeutic drugs can Selectively inhibits the growth of chemotherapy-resistant small cell carcinoma, especially chemotherapy-resistant small cell carcinoma with high expression of GGPS1; the present invention discloses for the first time a new therapeutic target of a class of chemotherapy-resistant small cell lung cancer and its application
  • a method for screening a substance for inhibiting chemotherapy-resistant small cell lung cancer comprising: (1) combining a candidate substance with GGPS1 (GGPP synthase)/RAB7A (Ras-related protein 7A) )/autophagy flux signaling pathway system contact; (2) screen out substances that regulate GGPS1/RAB7A/autophagy flux signaling pathway, and the substances are useful substances (including potential substances) for inhibiting chemotherapy-resistant small cell lung cancer;
  • the regulation includes: inhibiting the expression or activity of GGPS1, inhibiting the membrane localization of RAB7A, inhibiting the modification effect of GGPS1 metabolite GGPP on RAB7A, or promoting autophagic flow disorder.
  • the GGPS1/RAB7A/autophagy flux signaling pathway is included in the mevalonate pathway, or is a downstream pathway of the mevalonate pathway.
  • the GGPS1/RAB7A/autophagic flux signaling pathway includes: GGPS1 protein, RAB7A protein; the autophagic flux is autophagic flux triggered by the fusion of autophagosome and lysosome.
  • step (1) includes: adding candidate substances to the system containing the GGPS1/RAB7A/autophagy flow signaling pathway;
  • step (2) includes: detecting each protein in the GGPS1/RAB7A/autophagy flow signaling pathway Changes in the gene or its encoding gene, and compared with the control group, wherein the control group is a system containing the GGPS1/RAB7A/autophagy flow signaling pathway without adding the candidate substance; if the candidate substance inhibits the expression or activity of GGPS1 , inhibiting the membrane localization of RAB7A, inhibiting the modification of RAB7A by GGPS1 metabolite GGPP, or promoting autophagy flow disorder, the candidate substance is a useful substance for inhibiting chemotherapy-resistant small cell lung cancer.
  • the system containing the GGPS1/RAB7A/autophagy flow signaling pathway is selected from: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system or an animal system.
  • the improvement or promotion is a statistical improvement or promotion, for example, compared with the control or the base, the improvement or promotion is more than 10% or 20%, preferably 40% or 50%. % or more, more preferably 80% or 100% or more.
  • the inhibition can also be referred to as down-regulation, which is a statistical inhibition or down-regulation, such as inhibiting or down-regulation by more than 10% or 20% compared with the control or the base, preferably inhibiting or down-regulation. Down 40% or 50% or more, more preferably 80% or 100% or more.
  • the candidate substances include (but are not limited to): regulatory molecules (such as but Without limitation, upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (eg, antibodies or ligands), CRISPR constructs, small molecule compounds, compounds from compound libraries.
  • regulatory molecules such as but Without limitation, upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (eg, antibodies or ligands), CRISPR constructs, small molecule compounds, compounds from compound libraries.
  • the use of the GGPS1/RAB7A/autophagy flow signaling pathway is provided for screening substances that inhibit chemotherapy-resistant small cell lung cancer; preferably, the GGPS1/RAB7A/autophagic flow signaling pathway It is included in the mevalonate pathway, or is a downstream pathway of the mevalonate pathway; or, the GGPS1/RAB7A/autophagy flow signaling pathway includes: GGPS1 protein, RAB7A protein; the autophagy flow is autophagy. Autophagic flux triggered by the fusion of phagosomes and lysosomes.
  • a regulator that modulates the GGPS1/RAB7A/autophagy flow signaling pathway for the preparation of a pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer; wherein, the regulator comprises a compound selected from the group consisting of Lower panel: inhibitors of GGPS1 expression or activity, inhibitors of RAB7A membrane localization, inhibitors of the modification of RAB7A by GGPS1 metabolite GGPP, or promoters that promote disorders of autophagic flux.
  • the GGPS1 expression or activity inhibitor includes (but is not limited to): an agent for knocking out or silencing GGPS1 gene, an agent for inhibiting GGPS1 protein activity; preferably, including: specifically interfering with GGPS1 gene expression A CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent targeting the GGPS1 gene, the reagent mutates GGPS1 with loss-of-function.
  • the RAB7A membrane localization inhibitor includes (but is not limited to): an agent that knocks out or silences the RAB7A gene, and an agent that inhibits the activity of the RAB7A protein; preferably, it includes: specifically interferes with the expression of the RAB7A gene Interfering molecules, CRISPR gene editing reagents, homologous recombination reagents, or site-directed mutagenesis reagents targeting the RAB7A gene that mutate RAB7A loss-of-function.
  • the inhibitor of the modification effect of the GGPS1 metabolite GGPP on RAB7A includes (but is not limited to): an agent that inhibits the synthesis of GGPP by GGPS1.
  • the promoters promoting autophagic flow disorder include (but are not limited to): inhibitors of GGPS1 expression or activity, inhibitors of RAB7A membrane localization, inhibitors of modification of RAB7A by GGPS1 metabolite GGPP .
  • the reagent for knocking out or silencing the GGPS1 gene is a shRNA plasmid for knocking down GGPS1; the functional sequence for knocking down is: C CTGAGCTAGTAGCCTTAGTA
  • the reagent for knocking out or silencing the RAB7A gene is knock-down Low RAB7A shRNA plasmid; the functional sequence for knockdown is: GGCTAGTCACAATGCAGATAT.
  • a pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer comprising statins, etoposide and cisplatin.
  • kits for inhibiting chemotherapy-resistant small cell lung cancer which includes the pharmaceutical composition; or a container, and the statins, etiology, etc. respectively placed in the container. Poside and Cisplatin.
  • statins include Mevastatin, Simvastatin, PitvaStatin, Atorvastatin, LovaStatin. , FluaStatin.
  • the chemotherapy-resistant small cell lung cancer is a small cell lung cancer with high expression of GGPS1 resistance.
  • the "high expression” is “high expression” in a statistical sense, for example, "small cell lung cancer with high expression of GGPS1" and the total population of "small cell lung cancer” (or statistical significance) A sufficient amount of the population) is significantly higher than the average GGPS1 expression of 10% or 20%, preferably 30% or 50% higher, more preferably 80% or 100% higher.
  • the dosage ratio of the statins, etoposide and cisplatin is (35-105):(3-9):1; preferably ( 49 to 70): (4.5 to 7.5): 1 (eg 58.3: 5: 1).
  • the method of administration of the cisplatin, etoposide and the statin is as follows: 0.5-10 mg is administered on the first day by intraperitoneal injection according to the body weight of the individual, calculated as one course of treatment per week.
  • /kg eg 0.8-6 mg/kg, more specifically such as 1, 2, 3, 4, 5 mg/kg) cisplatin (CDDP); 0.5-15 mg/kg/day (eg 1 to 3 days) ⁇ 12 mg/kg/day, more specifically 2, 4, 6, 8, 10 mg/kg/day) etoposide (VP16); concurrently 2 to 100 mg/kg/day (eg 5 mg/kg/day) by gavage ⁇ 60 mg/kg/day, more specifically such as 6, 8, 10, 15, 20, 30, 40, 50 mg/kg/day) of mevastatin or simvastatin or pitavastatin and other drug treatments; preferably Typically, when the drug is placed in a kit, the method of administration is described in the instructions for use of the kit.
  • GGPS1 protein or its encoding gene in preparing a diagnostic reagent for diagnosing or prognosing small cell lung cancer; preferably, the diagnosing or prognosticating comprises: : According to the expression of GGPS1 protein, determine whether it is suitable for statin therapy; if GGPS1 protein is highly expressed, this therapy is applicable.
  • a reagent that specifically recognizes GGPS1 protein or its encoding gene for preparing a diagnostic reagent or a diagnostic kit for diagnosing or prognosing small cell lung cancer;
  • prognosis includes: according to the expression of GGPS1 protein, judging whether it is suitable for the treatment plan of statin; if the GGPS1 protein is highly expressed, the treatment plan is applicable.
  • the treatment regimen is a treatment regimen of a combination of statins, etoposide and cisplatin.
  • the diagnostic reagent comprises a primer selected from the group consisting of: a primer that specifically amplifies the gene encoding GGPS1 protein; a probe that specifically recognizes the gene encoding GGPS1 protein or its transcript; or a specific anti-GGPS1 protein of antibodies.
  • kits for diagnosing or prognosing small cell lung cancer contains: a diagnostic reagent for detecting the expression or expression level of GGPS1 protein or its encoding gene.
  • the kit further includes: nucleic acid extraction reagents, polymerase chain reaction reagents, western blotting reagents, and/or enzyme chain immunoreaction reagents.
  • Figure 1 Scheme for establishing chemotherapy-resistant tumors in mouse small cell lung cancer.
  • Figure 2 A mouse model of small cell lung cancer chemotherapy-resistant tumors.
  • IC50 of H82 and H82R cells was detected by detecting cell viability under E/P treatment.
  • Figure 4 Drug library screening with FDA-approved metabolites.
  • A The flow of drug library screening using H82 and H82R cells. There are 256 small molecule inhibitors used for drug library screening. Each well was plated with 2000 cells, and after 24 hours, the drug library was added at a concentration of 5 micromolar, and the cell viability was detected on the fourth day.
  • H82 and H82R cell growth was detected after cells were treated with MevaStatin at 2 ⁇ M, 5 ⁇ M and 10 ⁇ M for 72 hours.
  • H82 and H82R cells were examined after cells were treated with different concentrations of statins (AtorvStatin, PitvaStatin, LovaStatin, MevaStatin, SimvaStatin and FluaStatin).
  • statins AtorvStatin, PitvaStatin, LovaStatin, MevaStatin, SimvaStatin and FluaStatin.
  • Chemosensitive cell lines H146, H209 and H526
  • chemoresistant cell lines H196, H446 and DMS114
  • H82R cells were divided into four groups after subcutaneous tumor formation: control treatment, E/P treatment, MevaStatin treatment and combination treatment
  • Tumor weight graph for each group (middle), and mouse weight graph for each group (right).
  • D Statistical graph of the staining ratio of CC3 and H2AX in each group of tumors for H82R.
  • E.H82R cells were divided into multiple groups according to control treatment, E/P treatment, SimvaStatin treatment, PitvaStatin treatment and combined treatment.
  • the graphs show a graph of tumor growth in each group (left), a graph of tumor weight in each group (middle), and a graph of mouse body weight in each group (right).
  • MVA mevalonic acid
  • GGPP geranylgeranyl pyrophosphate
  • GGOH geranylgeraniol
  • FPP farnesyl pyrophosphate
  • FFOH farnesol
  • SQL squalene
  • CoQ9 coenzyme Q9
  • CoQ10 coenzyme Q10
  • Statin inhibits H82R cells by inhibiting GGPP production and affecting the intracellular distribution of RAB7A.
  • ATP1A1 sodium/potassium ion transporting ATPase ⁇ 1 peptide
  • Tubulin is an internal reference protein. All data analyses were performed using two-way ANOVA or unpaired t-test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 and ****p ⁇ 0.0001. All errors are standard error of the mean.
  • Statin causes autophagy disorder in H82R cells through GGPP-RAB7A-autophagy pathway.
  • H82 and H82R cells were treated with control, 5 ⁇ M MevaStatin, 5 ⁇ M MevaStatin and 2 ⁇ M GGPP for 72 hours, and the expression levels of P62 and LC3B were detected by WB.
  • H82 and H82R cells were treated with control, 5 ⁇ M MevaStatin, 5 ⁇ M MevaStatin and 2 ⁇ M GGPP for 72 hours, and the expression levels of PARP and CC3 were detected by WB.
  • Sensitive cell lines H209, H526) and resistant cell lines (H446, DMS114) cells were treated with control and 5 ⁇ M MevaStatin for 72 hours, and the expression levels of P62 and LC3B were detected by WB.
  • WB detected the expression levels of P62 and LC3B in H82R tumor control group, MevaStatin group and combined treatment group.
  • the number of autophagolysosomes in the cells was counted after the stably transfected cell line H82R mRFP-GFP-LC3 was treated with control, 5 ⁇ M MevaStatin, 5 ⁇ M MevaStatin and 2 ⁇ M GGPP for 72 hours.
  • mice were divided into four groups, which were treated with control, E/P, MevaStatin and combined treatment. Tumor volume was calculated by vernier caliper measurement. Graphs of tumor growth curves for each group (left) and tumor weights for each group (right) are shown.
  • FIG. 14 Statin inhibits PDX in PD small cell lung cancer with high expression of GGPS1.
  • GGPS1 geranylgeranyl diphosphate synthase 1
  • the present invention discloses for the first time a novel signaling pathway closely related to the diagnosis and treatment of small cell lung cancer: GGPS1/RAB7A/autophagy flow signaling pathway.
  • the invention discloses a new mechanism involved in the signaling pathway to regulate small cell lung cancer and its drug resistance.
  • small cell lung cancer cell lines (referred to as H82, H209, H526 and H146 in the examples) are sensitive to chemotherapeutic drugs (eg E/P) both in vitro and in vivo.
  • chemotherapeutic drugs eg E/P
  • H82R, H209R, H526R and H146R chemotherapy-resistant tumors
  • the method for the drug-resistant small cell lung cancer cell line comprises: (a) transplanting small cell lung cancer cells sensitive to chemotherapeutic drugs into a recipient; (b) the recipient of (a) Administering chemotherapeutic drug treatment to make the tumor formed by the chemotherapeutic drug-sensitive small cell lung cancer cells resistant; preferably, the treatment includes: after the chemotherapeutic drug-sensitive small cell lung cancer cells are administered to the receptor, the tumor volume grows to about 50-400mm 3 (preferably, 100-200mm 3 ), give etoposide and cisplatin (E/P) chemotherapy drugs; if the tumor subsides significantly after administration, the administration should be suspended, and the tumor will resume growth again. Continue the administration; repeat this process until the chemotherapeutic drugs cannot effectively inhibit tumor growth; (c) isolate small cell lung cancer cells from the tumor of the recipient of (b), carry out primary culture and/or subculture, and acquire drug resistance Small cell lung cancer cell lines.
  • E/P etoposide and cisplatin
  • the drug library was screened using FDA-certified metabolic drugs, and it was found that statins can significantly inhibit the survival of chemotherapy-resistant small cell lung cancer.
  • the "(signal) pathway” refers to a signal system formed by mutual restriction or interaction between a series of genes or proteins or their metabolites (synthetic products or processing products), and also includes pathways The interaction of a protein with other elements or organelles in the cell, sometimes including the joint participation of its upstream and downstream genes or proteins, generally leads to the occurrence of some cellular events.
  • the GGPS1/RAB7A/autophagy flux signaling pathway mainly includes the following elements: GGPS1 gene (and/or its encoded protein), RAB7A gene (and/or its encoded protein), and autophagy flux signaling pathway.
  • the autophagic flow signaling pathway includes the participation of autophagosome and lysosome.
  • GGPS1/RAB7A/autophagic flux signaling pathway and "GGPS1-RAB7A-autophagic flux signaling pathway” are used interchangeably.
  • the nucleotide sequence of the GGPS1 gene is shown in, for example, GenBank_NC_000001.11 (human origin); the protein amino acid sequence thereof is shown in, for example, GenBank_AAH67768.1 (human origin).
  • the nucleotide sequence of the RAB7A gene is shown, for example, in GenBank_NC_000003.12 (human origin); the protein amino acid sequence thereof is shown in, for example, GenBank_AAH13728.2 (human origin).
  • the nucleotide sequence of the HMGCR gene is shown, for example, in GenBank_NC_000005.10 (human origin); the protein amino acid sequence thereof is shown in, for example, GenBank_AAH33692.1 (human origin).
  • the above-mentioned protein also includes its variant forms, including (but not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1- 10, still more preferably such as 1-8, 1-5) amino acid deletions, insertions and/or substitutions, and C-terminal and/or N-terminal additions or deletions of one or more (usually within 20, Preferably within 10, more preferably within 5) amino acids.
  • Any with high homology to the protein such as 70% or more homology to the polypeptide sequence; preferably 80% or more homology; more preferably 90% or more homology High, such as 95%, 98% or 99% homology
  • the present invention also includes a mutant form of the protein or protein truncate, so long as the mutant protein or truncate substantially retains the function of the full-length protein.
  • sequences of the above-mentioned genes also include degenerate sequences therewith.
  • Polynucleotides (genes) encoding proteins can be native genes or their degenerate sequences.
  • statins can significantly inhibit the survival of chemotherapy-resistant tumors. Further in vitro and in vivo experiments confirmed that Statin can inhibit the growth of chemotherapy-resistant cells in multiple small cell lung cancers. The inventors found that the downstream metabolite GGPP of the mevalonate pathway can completely reverse the inhibitory effect of Statin on drug-resistant cells.
  • the MVA pathway is a metabolic pathway for the synthesis of isoprene pyrophosphate and dimethylallyl pyrophosphate from acetyl-CoA, which exists in all higher eukaryotes and many viruses.
  • the products of this pathway are the synthetic precursors of biomolecules such as steroids and terpenoids, and are also necessary steps in the cholesterol synthesis pathway. Its downstream is involved in many important functions of cells, such as cholesterol synthesis, mitochondrial function, and protein prenylation modification.
  • HMGCR is the initial and rate-limiting step of the MVA metabolic pathway, which synthesizes mevalonate, a metabolic enzyme that can be inhibited by statins.
  • FPP will be synthesized by FDPS
  • FPP will be synthesized into squalene through FDFT1
  • squalene will continue to synthesize cholesterol by SQLE.
  • FPP can also synthesize GGPP through GGPS1, and GGPP can carry out geranylgeranylation modification of target protein, which belongs to isoprene modification and plays an important function in cells.
  • statins mainly inhibit the production of GGPP in the MVA metabolic pathway, interfering with the geranylgeranylation modification of the small G protein RAB7A by GGPP, so that the latter cannot perform normal functions on the membrane. This in turn inhibits the fusion of autophagosomes and lysosomes, triggering autophagic flux barriers, and ultimately leading to the death of chemotherapy-resistant cells. That is, Statin is mediated by the GGPS1-RAB7A-Autophagic pathway (autophagic flow signaling pathway), which can lead to autophagic flow barriers, trigger cell death, and inhibit the survival of drug-resistant cancer cells.
  • GGPS1-RAB7A-Autophagic pathway autophagic flow signaling pathway
  • the "inhibitor (agent)” and “down-regulating (agent)” can be used interchangeably, which also include: block (agent), antagonist (agent) and the like.
  • GGPS1/RAB7A/autophagy flow signaling pathway preferably, also including its upstream and downstream proteins or genes
  • various methods well known to those in the art can be used to regulate the The GGPS1/RAB7A/autophagic flow signaling pathway.
  • various methods well known to those skilled in the art can be used to modulate or deplete the expression of pathway proteins.
  • use methods well known to those skilled in the art to promote or attenuate the occurrence of autophagic flux can be used to regulate the The GGPS1/RAB7A/autophagic flow signaling pathway.
  • a down-regulating agent that down-regulates the expression or activity of GGPS1, down-regulates the membrane localization of RAB7A, or down-regulates the modification effect of GGPS1 metabolite GGPP on RAB7A.
  • the down-regulating agent refers to any substance that can reduce the activity of GGPS1 or RAB7A, reduce the stability of GGPS1 or RAB7A, down-regulate the expression of GGPS1 or RAB7A, reduce the effective time of GGPS1 or RAB7A, and inhibit the transcription and translation of GGPS1 or RAB7A. , these substances can be used in the present invention as potentially useful substances for inhibiting small cell lung cancer.
  • the biomolecules can be chemical compounds, small chemical molecules, biomolecules.
  • the biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.
  • the down-regulating agents are: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of GGPS1 or RAB7A or their upstream genes; or specifically editing GGPS1 or RAB7A or their upstream gene editing agents, and so on.
  • the present invention provides a method for down-regulating the GGPS1/RAB7A/autophagy flow signal pathway, including targeted mutation, gene editing or gene recombination of the GGPS1 or RAB7A gene in the GGPS1/RAB7A/autophagy flow signal pathway, so as to achieve down.
  • GGPS1 or RAB7A can be converted into a loss-of-function truncation or mutant thereof by any of the above methods.
  • the CRISPR/Cas system is used for gene editing, thereby knocking out or down-regulating the target gene. Appropriate sgRNA target sites will bring higher gene editing efficiency, so before proceeding with gene editing, suitable target sites can be designed and found. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments.
  • a method for down-regulating the expression of GGPS1 or RAB7A comprising: transferring an interfering molecule that interferes with the expression of GGPS1 or RAB7A into cells, or treating cells by appropriate means to make them Introduced into cells, such as designing the transmembrane domain to have the ability to transmembrane.
  • the above proteins or coding genes may be naturally occurring, for example, they may be purified and isolated from mammals; they may also be recombinantly prepared, for example, according to conventional genetic recombination technology to produce recombinant proteins.
  • any variation that does not affect the biological activity of these proteins is available, such as derivatives or variants that do not alter their function.
  • the research on the GGPS1/RAB7A/autophagy flow signaling pathway has various uses, and the uses include: screening for substances that regulate this signaling pathway, in order to inhibit small cell lung cancer.
  • the regulation includes: down-regulating the expression or activity of GGPS1, down-regulating the membrane localization of RAB7A, down-regulating the modification effect of GGPS1 metabolite GGPP on RAB7A, or promoting autophagic flow disorder, etc.
  • the present invention provides a method for screening regulators regulating GGPS1/RAB7A/autophagy flow signaling pathway, by adding candidates to be screened to a system containing GGPS1/RAB7A/autophagy flow signaling pathway, and observing GGPS1/RAB7A/ The changes or interactions of each protein or gene in the autophagic flow signaling pathway were screened. If the candidate substance down-regulates the expression or activity of GGPS1, down-regulates the membrane localization of RAB7A, down-regulates the modification effect of GGPS1 metabolite GGPP on RAB7A, or promotes the disturbance of autophagic flow, the candidate substance is a useful substance for inhibiting small cell lung cancer.
  • the terms “inhibit”, “increase” and “promote” all refer to “inhibit”, “increase” and “promote” with statistical significance. That is: significantly “inhibit”, “increase”, “promote”. For example, compared with the protein activity, protein expression, protein binding or methylation degree of the control group, significantly “inhibit”, “increase”, “promote” 10%, 20%, 30%, 40%, 50% or more; more Best 60%, 70%, 80% or more.
  • the system containing GGPS1/RAB7A/autophagy flow signaling pathway is selected from: cell system (or cell culture system), subcellular system (or subcellular culture system), solution system, animal system or tissue system (or tissue culture system).
  • the method further includes: performing further cell experiments and/or animal experiments on the obtained potential substances to further select and determine the substances useful for inhibiting small cell lung cancer from the candidate substances.
  • a variety of conventional techniques can be used to identify transcription or expression of genes in the system. These techniques include, but are not limited to, oligonucleotide hybridization techniques (eg, probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and the like.
  • oligonucleotide hybridization techniques eg, probes
  • PCR polymerase chain reaction
  • polyacrylamide gel electrophoresis and the like.
  • co-immunoprecipitation technique eg.g, probes
  • PCR polymerase chain reaction
  • nuclear localization of proteins is also a technique well known in the art.
  • Substances preliminarily screened by the above method can constitute a screening library, so that people can finally screen out substances that are really useful for inhibiting small cell lung cancer.
  • the present invention also provides potential substances obtained by the screening method which can be used to inhibit small cell lung cancer.
  • the present invention also provides a method for preparing a drug for inhibiting small cell lung cancer (especially for inhibiting small cell lung cancer), the method comprising: synthesizing and/or purifying the substances obtained by the aforementioned screening that are useful for inhibiting small cell lung cancer, as Drugs for the inhibition of small cell lung cancer.
  • the obtained substances useful for inhibiting small cell lung cancer can be used for the preparation of pharmaceutical compositions, as hereinafter described in the present invention.
  • the methods for screening substances acting on the target by taking a protein or gene or a specific region on it as a target are well known to those skilled in the art, and these methods can be used in the present invention.
  • the candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the type of substances to be screened, it is clear to those skilled in the art how to select a suitable screening method.
  • statins inhibit the production of GGPP in the MVA metabolic pathway, interfere with the geranylgeranylation modification of the small G protein RAB7A by GGPP, and make the later
  • the cells are unable to function normally on the upper membrane, which in turn inhibits the fusion of autophagosomes and lysosomes, triggers autophagic flow barriers, and eventually leads to the death of chemotherapy-resistant cells.
  • down-regulating GGPS1 can further reduce the geranylgeranylation modification of small G protein RAB7A by GGPP, or directly down-regulate RAB7A, thereby inhibiting the fusion of autophagosome and lysosome, and causing autophagic flow disorder .
  • the present invention provides a pharmaceutical composition for inhibiting small cell lung cancer, including a drug combination: statins, etoposide and cisplatin.
  • inhibiting small cell lung cancer includes “reducing (reversing) the drug resistance of small cell lung cancer”.
  • the small cell lung cancer is drug-resistant small cell lung cancer.
  • the term "effective amount” or “effective dose” refers to that which is functional or active in humans and/or animals and which is acceptable to humans and/or animals as used herein.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the present invention also provides a kit for inhibiting tumor or reducing drug resistance of tumor, the kit includes statins, etoposide and cisplatin. More preferably, the kit also includes: instructions for use to guide the clinician to use the medicine in a correct and reasonable manner.
  • statin, etoposide, and cisplatin are combined and placed in a kit in unit dosage form.
  • Unit dosage form refers to the preparation of a drug into a single dosage form for the convenience of taking, including but not limited to various solid dosage forms (eg, tablets), liquid dosage forms, capsules, and sustained-release dosage forms.
  • statins, etoposide and cisplatin can also be separately placed in different containers, mixed when necessary, and applied.
  • the dosage ratio of the statins, etoposide and cisplatin is (5-15):(1-3):1; preferably (7-10):(1.5) ⁇ 2.5): 1 (eg 8.6: 1.7: 1).
  • the method of administration of the cisplatin, etoposide and the statin is as follows: one course of treatment per week, according to individual body weight, 0.5-10 mg is given by intraperitoneal injection on the first day /kg (eg 0.8-6 mg/kg, more specifically such as 1, 2, 3, 4, 5 mg/kg) cisplatin (CDDP); 0.5-15 mg/kg/day (eg 1 to 3 days) ⁇ 12 mg/kg/day, more specifically 2, 4, 6, 8, 10 mg/kg/day) etoposide (VP16); concurrently 2 to 100 mg/kg/day (eg 5 mg/kg/day) by gavage ⁇ 60 mg/kg/day, more specifically such as 6, 8, 10, 15, 20, 30, 40, 50 mg/kg/day) of mevastatin or simvastatin or pitavastatin and other drug treatments.
  • the individual is a mouse, and according to the calculation of one course of treatment per week, etoposide (VP16) is administered at 10 mg/kg/day on the 1st, 2nd, and 3rd days according to the body weight of the mouse.
  • etoposide (VP16) is administered at 10 mg/kg/day on the 1st, 2nd, and 3rd days according to the body weight of the mouse.
  • Cisplatin (CDDP) was administered at 6 mg/kg/day on day 1
  • drug treatment was administered by intraperitoneal injection.
  • Statins were administered by gavage at 50 mg/kg/day according to the body weight of mice, such as mevastatin, simvastatin, pitavastatin and other drugs.
  • A is the body surface area, calculated in m2 ; W is the body weight, calculated in g; K is a constant, which varies with animal species. Dog 11.2, Monkey 11.8, Human 10.6.
  • the conversion of the administered dose may vary according to the assessment of an experienced pharmacist.
  • GGPS1 may serve as a potential molecular marker to guide the administration of Statin in drug-resistant patients.
  • the inventors also used TCGA data to analyze the relationship between GGPS1 expression in clinical samples and patient prognosis, and found that patients with high expression of GGPS1 had a worse prognosis.
  • GGPS1 can be used as a marker for the diagnosis or prognosis of small cell lung cancer, especially as a marker for prognostic assessment in the post-chemotherapy stage: (i) Disease classification in the post-chemotherapy stage of cancer , differential diagnosis, and/or analysis of disease-free survival; (ii) evaluation of tumor treatment drugs, drug efficacy, prognosis, and selection of appropriate treatment methods in relevant populations. For example, people with abnormal GGPS1 gene expression can be isolated, allowing for more targeted treatment.
  • GGPS1 By judging the expression or activity of GGPS1 in the sample to be evaluated, the disease prognosis of the subject who provides the sample to be evaluated can be predicted, and an appropriate drug can be selected for treatment.
  • a threshold value of GGPS1 can be specified, and when the expression of GGPS1 is higher than the specified threshold value, a regimen of suppressing GGPS1 should be considered for treatment.
  • the threshold can be easily determined by those skilled in the art.
  • the threshold for abnormal GGPS1 expression can be obtained by comparing and analyzing the general GGPS1 expression in patients with small cell lung cancer or the expression in normal healthy people.
  • the present invention provides the use of the GGPS1 gene or protein for preparing a reagent or kit for evaluating the prognosis of small cell lung cancer.
  • the presence or absence and expression of GGPS1 gene or protein can be detected by various techniques known in the art, and these techniques are all included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination.
  • the present invention also provides reagents for detecting the presence or absence and expression of the GGPS1 gene or protein in an analyte.
  • primers that specifically amplify GGPS1 can be used; or a probe that specifically recognizes GGPS1 can be used to determine the presence or absence of the GGPS1 gene; when detecting the protein level, specific primers can be used.
  • the expression of GGPS1 protein is determined by binding the antibody or ligand of GGPS1 protein.
  • the kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, display solution, etc. Color solution, lotion, etc.
  • the kit may also include instructions for use and/or nucleic acid sequence analysis software, and the like.
  • DH5 ⁇ Competent E.coli
  • Plasmid Mini Kit Generay
  • Plasmid Intermediate Kit TIANGEN
  • DMEM basal medium Cat. No. SH30243.01B
  • RPMI 1640 basal medium Cat. No. SH30809.01B
  • FBS Fetal Bovine Serum
  • S0615 500ml/bottle, purchased from Biochrom AG, Germany, 40ml/tube and stored at -20°C
  • Penicillin, Streptomycin storage solution Item No. : 15140122, 100ml/bottle, purchased from Invitrogen company
  • RevertAidTM First Strand cDNA Synthesis Kit purchased from U.S.
  • pLKO.1-U6-puro was purchased from Addgene. Firstly, the vector is digested with AgeI/EcoRI double enzyme, and the 21-nucleotide sequence in the 5'-UTR, CDS or 3'-UTR sequence of the target gene is added to the XhoI restriction site and its reverse complement is added. inserted into the vector.
  • Lentiviral packaging plasmids PSPA and PMD2.G.
  • Retroviral packaging plasmid pCL10A. The pBabe-mCherry-GFP-Puro plasmid was provided by Memorial Sloan Kettering Cancer Center.
  • pLKO.1-U6-Tet-on-puro was purchased from Addgene and can be knocked down conditionally. Minimal extraction of plasmids (TIANGEN, DP107) and large-scale preparation of plasmids (TIANGEN, DP117) were accomplished using kits.
  • the shRNA sequence used to knock down GGPS1 was C CTGAGCTAGTAGCCTTAGTA :
  • the primer sequence is shhGGPS1-CDS1-F:
  • the shRNA sequence for knocking down RAB7A is: ggctagtcacaatgcagatat (SEQ ID NO:3);
  • the primer sequence is shhRAB7A-CDS1-F:
  • Small cell lung cancer cell lines H146, H196, DMS114 cells purchased from ATCC, culture conditions RPMI medium (Hyclone)+8%FBS+1 ⁇ P/S.
  • Small cell lung cancer cell lines H446 and H526 cells were provided by Shanghai Jiaotong University, and the culture conditions were RPMI (Hyclone) ligand + 8% FBS + 1 ⁇ P/S.
  • Adherent cells were taken out and the density was observed under an inverted microscope. Generally, the density greater than 70% was suitable for passage.
  • first aspirate the culture medium add 2mL of 1 ⁇ PBS (10cm Dish) to wash twice, then add trypsin digestion solution in an amount of 1ml/ 25cm2 area, shake up and down, left and right to evenly cover the cells, put in 37 °C incubator digestion, take out the gap and observe the cells become rounded under the microscope, then add an appropriate amount of medium to terminate the digestion, blow the cells to single cells with a 1mL pipette, and follow a certain ratio (usually 1:3 or 1:4). ) into a new petri dish. Shake cells in a cross shape and place in a 37°C incubator.
  • the target gene is prepared into retrovirus (retrovirus) or lentivirus (lentivirus) carrying exogenous target gene by calcium phosphate method.
  • retrovirus retrovirus
  • lentivirus lentivirus
  • Volume of 4mg/ml polybrane liquid (1:1000 dilution, final concentration 4 ⁇ g/ml) mix well, pipette an appropriate amount of virus liquid into the medium of the cells to be infected, and cultivate in a 37°C incubator. After 24 hours, replace with fresh virus solution and continue to infect cells.
  • viruses carrying antibiotic genes add corresponding antibiotics to screen for positive clones (Puromycin or G418, different cells are screened with different concentrations), and screen for 5-7 days;
  • Viruses with fluorescently labeled genes were screened with corresponding fluorescent markers (copGFP or DsRed2). Collect total RNA and total protein of virus-infected and screened cells, and perform real-time RT-PCR and western blot to detect the expression of target gene transcription and translation levels in these cells to identify overexpressed exogenous genes or shRNA silencing Establishment of stable cell lines with endogenous genes.
  • the H82R pBabe-mCherry-GFP-LC3 stable transfection cell line H446pBabe-mCherry-GFP-LC3 stable cell line, H82R pLko.1-Tet-shGGPS1 stable cell line, pLko.1- Tet-shRAB7A stably transfected cell line.
  • Antibodies used are listed in Table 1.
  • PDX human-derived small cell lung cancer tumor model
  • the PDX is considered to be established successfully.
  • a 100 ⁇ L volume was resuspended in sterile 1 ⁇ PBS.
  • the cell suspension was subcutaneously injected into the back sides of the nude mice or the armpits of the forelimbs.
  • tissue inoculate a tissue block size of approximately 3x3 mm subcutaneously on the back of the mouse.
  • tissue volume length ⁇ width ⁇ width/2
  • the mice were removed and the subcutaneous tumor weight was weighed. After being fixed overnight, paraffin embedding, sectioning and H&E staining were performed.
  • Immunohistochemical LSAB method (Labelled StreptAvidin-Biotin, streptavidin-biotin labeling method) was used to detect the expression of target protein in tissue samples.
  • Sample preparation Discard the culture medium of the adherent cultured cell lines (suspend the cells by centrifugation to remove the supernatant), rinse with PBS twice, and try to absorb the remaining PBS solution, add 100-200 ⁇ l 1 ⁇ SDS protein lysis buffer to each bottle, Gently scrape the cells with a cell scraper and collect them into a 1.5ml EP tube. After the lysis is complete, use a 1ml syringe to pipette the viscous material up and down until the lysate turns into a hanging drop. Save for later use; BCA (standard curve) protein quantification. The target protein was detected by Western Blot. Cell membrane protein and cytoplasmic protein were extracted using Biyuntian (P0033) kit.
  • E/P drugs When establishing a chemotherapy resistance model, when the tumor reaches the dosed volume, the mice are treated with E/P drugs, calculated as a course of 1 week, and 10 mg/kg/day according to the body weight of the mice. , Etoposide (VP16) was administered on days 2 and 3, and cisplatin (CDDP) was administered on day 1 at 6 mg/kg/day, diluted with medicinal saline, and the final injection volume was 0.2 mL/mouse, Intraperitoneal injection. Etoposide was purchased from Sigma, Cat. No. E1383. Cisplatin was purchased from Sigma, Cat. No. P4394. Stock concentrations were formulated with DMSO (dimethyl sulfoxide). Etoposide is 30 mg/ml and cisplatin is 1.5 mg/ml.
  • DMSO dimethyl sulfoxide
  • Statin drug When the tumor reaches the dosed volume, the mice are treated with Statin drug, administered daily, according to the body weight of the mice, 50 mg/kg/day of mevastatin, simvastatin, pitavastatin and other drugs are administered by gavage . It was diluted with medicinal saline, and the final injection volume was 0.2 mL/mouse.
  • Mevastatin was purchased from MCE Company, catalog number HY-17408. Simvastatin was purchased from MCE Company, product number HY-17502, and pitavastatin was purchased from MCE Company, product number HY-B0165A.
  • Drug library screening experiment The cells to be treated are plated in a 96-well plate according to 2500 cells per well, and the volume of each well is 100 microliters. After 24 hours, the drug to be treated is prepared according to the 2X concentration. The drug was added to the 96-well plate in a volume of 100 microliters per well, with a final volume of 200 microliters.
  • the information of the drug library used is the metabolic small molecule inhibitor certified by the FDA of MCE Company, the product number is HYCPK4390, HYCPK4391, HYCPK4392, HYCPK4393. After 72 hours, CTG reagent was added for detection. Drug library screening data were analyzed based on reads.
  • Stable transfection cell lines H82R pBabe-mCherry-GFP-LC3, H446pBabe-mCherry-GFP-LC3 and DMS114pBabe-mCherry-GFP-LC3 were established according to the previously mentioned plasmids, and the cells were plated in 12-well plates, and coverslips had been added in advance piece. 24h later:
  • Gene knockdown experiment lentivirus infected cells knock down RAB7A and GGPS1. 3 days After treatment, cells were collected and examined under a microscope.
  • autophagolysosomes are normally formed as yellow dots, but when autophagic flow is blocked, autophagosomes are normally formed, but cannot combine with lysosomes to form autophagolysosomes, so For red dots.
  • the inventors used four chemosensitive cell lines (H82, H209, H526 and H146) and two chemoresistant cell lines (H446 and DMS114), according to the The number of cells at 1 ⁇ 10 6 / mouse was subcutaneously injected into the back of nude mice, and the tumor volume grew to about 100-200 mm 3 , and divided into two groups: the control group was treated with normal saline (Fig. 1A); SOP) were treated with etoposide and cisplatin (E/P) chemotherapeutic drugs (Fig. 1B).
  • mice inoculated with H82, H209 and H526 tumors had longer survival in the presence of chemotherapeutic drug treatment, while the survival of mice inoculated with drug-resistant tumors was shorter (Fig. 2B,C).
  • IHC detection showed that the expression of apoptosis marker CC3 and DNA damage marker H2AX increased, and apoptosis and DNA damage increased in sensitive tumors under drug treatment.
  • chemotherapy-resistant tumors showed no changes in CC3 and H2AX, and did not induce apoptosis and cause DNA damage (Fig. 2D). This suggests that chemotherapy-resistant tumors have not responded to chemotherapy drugs, and the chemotherapy-resistant model of small cell lung cancer was successfully established.
  • mice The inventors also inoculated the small cell lung cancer chemotherapy-resistant cell lines H446 and DMS114 in nude mice, and found that the tumors initially did not respond to chemotherapy drugs (Fig. 2E). Survival analysis showed that mice inoculated with H446 and DMS114 tumors had short survival in the presence of chemotherapeutic drug treatment (Fig. 2F,G).
  • H82R drug-resistant tumors
  • H209R primary small cell lung cancer chemotherapy-resistant cell lines
  • H526R primary small cell lung cancer chemotherapy-resistant cell lines
  • the inventors found that, compared with H82 cells, H82R cells exhibited high resistance to chemotherapeutic drugs (Fig. 3A, B); while H209R and H526R cells did not exhibit more significant chemoresistance than H209 and H526 cells (Fig. 3C, D).
  • the chemoresistance mechanism may be related to the tumor microenvironment, and the chemoresistance cell H82R is highly resistant to chemotherapeutic drug treatment in vivo and in vitro, which is more suitable for subsequent drug screening and mechanism research on cells.
  • H82R was resistant to E/P treatment alone, and cell growth was not affected under continuous administration treatment.
  • sensitive to Statin treatment and combined treatment of Statin plus E/P cell growth was significantly inhibited (Fig. 5D). This suggests that Statin can inhibit the growth of chemotherapy-resistant H82R cells.
  • H82R cells to subcutaneously form tumors, and then divided into 4 groups: control group, E/P treatment group, MevaStatin treatment group, E/P and MevaStatin combined treatment group.
  • control group E/P treatment group
  • MevaStatin treatment group E/P and MevaStatin combined treatment group.
  • the inhibition of small molecule drugs is often not specific enough, in order to clarify which specific downstream metabolites of the MVA pathway have important functions and effects on drug-resistant cells.
  • the present inventors supplemented the downstream metabolites of the MVA pathway at the same time in the case of administration of Statin.
  • the results showed that in addition to MVA could reverse the inhibitory effect of Statin on H82R cells, GGPP and GGOH could also reverse the inhibitory effect of Statin on H82R cells.
  • GGOH does not exist in cells, but has the same group as GGPP, and plays the role of protein geranylgeranylation (Fig. 8A).
  • Example 9 Statin inhibits the production of GGPP in H82R cells and affects the membrane localization of RAB7A
  • GGPP the downstream product of MVA, can reverse the inhibitory effect of Statin on drug-resistant cells, and the inventors want to study the mechanism by which GGPP exerts this effect.
  • GGPP mainly modifies small G protein (Small GTP-ase) through geranylgeranylation after protein translation, so that it binds to the membrane and is further phosphorylated by GTPase to function.
  • small G proteins Small GTP-ase
  • the inventors constructed a shRNA library to knock down the target gene in H82R cells.
  • the library selected important small G proteins (RHOA, RHOB, RAP1A, RAP1B) in cells , CDC42), and key metabolic enzyme genes in the MVA pathway.
  • Example 10 Knockdown of GGPS1 or RAB7A induces autophagy disorder in chemo-resistant cells
  • Autophagy lysosomes can remove damaged proteins and organelles in cells and provide cells with substances and energy. During the whole process, cells phagocytose the degraded substances into autophagosomes, and the process of forming and degrading substances in autophagolysosomes is called autophagic flux. Studies have shown that the association of autophagosomes and lysosomes requires RAB7A to help form autophagolysosomes. Inhibition of RAB7A function will affect the formation of autophagolysosomes and cause autophagic flow disorders. In order to determine the status of autophagic flux in H82R cells, the inventors intend to detect the protein expression levels of P62 and LC3B.
  • LC3B indicates the formation of intracellular autophagosomes.
  • P62 has substrate specificity. It connects LC3B and the ubiquitinated protein to be degraded into autophagosomes, and then fuses with lysosomes to form autophagolysosomes to be degraded, which can be used to indicate autophagic flow. Is it normal. When autophagic flux is normal, autophagosomes are increased, P62 is degraded, and its expression is decreased. If autophagolysosome formation is affected and autophagic flux is blocked, LC3B will increase, while P62 cannot be degraded and its expression level remains unchanged.
  • LC3B was significantly increased in chemotherapy-resistant cell lines (H446, DMS114), and P62 was not significantly changed after Statin treatment in other small cell lung cancer cells. This suggested that Statin could affect the formation of autophagolysosomes in drug-resistant cell lines, causing autophagic flux barriers (Fig. 10C).
  • RAB7A is critical for the formation of autophagic lysosomes.
  • the inventors knocked down GGPS1 or RAB7A in H82R, and then detected the expression of P62 and LC3B.
  • the results showed that knockdown of GGPS1 or RAB7A significantly increased intracellular LC3B, but no significant change in P62. This suggests that GGPS1 and RAB7A are key proteins that ensure smooth autophagic flow in chemotherapy-resistant cells (Fig. 10D).
  • the inventors also found that knockdown of GGPS1 or RAB7A induced apoptosis (Fig. 10E).
  • HMGCR as the initiation gene of the MVA pathway, knockdown of HMGCR in cells also caused a significant increase in LC3B and CC3 (Fig. 10F).
  • the inventors used the method of fluorescent labeling to detect the occurrence process of autophagy. After establishing H82R pBabe-mCherry-GFP-LC3 and H446pBabe-mCherry-GFP-LC3 stably transfected cell lines, the inventors treated the cells with Statin, and further observed the intracellular autophagic flow by immunofluorescence. The results showed that in the cells of the control group, the level of RFP+GFP-puncta indicating normal autophagic flux was high, and the level of RFP+GFP+puncta indicating impaired autophagic flux was low.
  • RFP+GFP-puncta was also found to be low, while RFP+GFP+puncta, which indicates a disorder of autophagic flow, was increased (FIG. 11C).
  • GGPS1 has a more obvious inhibitory effect on tumors than knockdown of RAB7A, suggesting that this is because GGPS1 is very important for cell function, and many small G proteins are affected, not just RAB7A.
  • Example 13 Statin inhibits the growth of chemotherapy-resistant PDX tumors in small cell lung cancer with high expression of GGPS1
  • FIG. 13A In order to determine the therapeutic effect of Statin on small cell lung cancer PDX tumors, the inventors established a small cell lung cancer chemotherapy-resistant PDX tumor mouse model ( FIG. 13A ).
  • the inventors found that the inoculated tumors were initially sensitive to E/P treatment, and subsequently treated with chemotherapy drugs in vivo, the tumors gradually became resistant until the Small cell lung cancer chemotherapy-resistant tumors PDX ZS7R and ZS4R (Fig. 13A, B) (ZS7R and ZS4R refer to drug resistance model code).
  • Growth curves and survival analysis of tumors showed that ZS7R and ZS4R were already resistant to E/P chemotherapeutic drug treatment relative to PDX tumors ZS7 and ZS4 ( Figure 13A,B).
  • the expression of MVA-related genes in ZS7, ZS7R and ZS4, ZS4R was detected by WB.
  • the results showed that FDPS and GGPS1 of the MVA pathway in ZS7R were highly expressed, while FDFT1 and SQLE were low expressed in ZS7R compared with sensitive tumors.
  • the MVA pathway in ZS4R has low expression of FDPS and GGPS1, but high expression of FDFT1 and SQLE, indicating that the MVA pathway is more GGPP synthesized in ZS7A (chemotherapy drug-sensitive model code), while ZS4R is more synthesized in cholesterol (FIG. 13C). This suggests that ZS7R is more suitable for Statin treatment than ZS4R.
  • Example 14 Statin inhibits the growth of chemotherapy-resistant PDX tumors in small cell lung cancer with high GGPS1 expression
  • PD Progressive disease, with an increase of at least ⁇ 20% in the sum of the largest diameters of target lesions, or the appearance of new lesions.
  • SD stable disease, the sum of the largest diameters of target lesions does not reduce to PR, or the increase does not reach PD.
  • PR Partial response, with a reduction of ⁇ 30% in the sum of the largest diameters of target lesions.
  • CR Complete response (complete response), the target lesions completely disappeared.
  • IHC and WB results showed that in 10 small cell lung cancer PDXs, GGPS1 was highly expressed in small cell lung cancer patients in SD and PD stages (Fig. 14A, B), and GGPS1 expression was highest in PD small cell lung cancer patients (Fig. 14A, B). 14A, B, C).
  • the inventors inoculated SP9 (tumor model code) of small cell lung cancer in PD stage, and grouped them into different drug treatments. The results showed that Statin and Statin plus E/P treatment significantly inhibited SP9 tumor growth.
  • Example 15 Analysis of TCGA database shows that patients with small cell lung cancer with high expression of GGPS1 have a worse prognosis

Abstract

Provided in the present invention are a new target for the diagnosis and treatment of chemotherapy drug-resistant small cell lung cancer and an application thereof. The present invention discloses a novel signal pathway closely related to the treatment of chemotherapy drug-resistant small cell lung cancer: the GGPS1/RAB7A/autophagy flow signal pathway; and discloses a new mechanism for regulating small cell lung cancer and the drug resistance thereof involving said signal pathway.

Description

诊断和治疗化疗耐药小细胞肺癌的新靶点及其应用New targets for diagnosis and treatment of chemotherapy-resistant small cell lung cancer and their applications 技术领域technical field
本发现属于分子生物学以及肿瘤学领域，更具体地，本发明涉及诊断和治疗化疗耐药小细胞肺癌的新靶点及其应用。The discovery belongs to the fields of molecular biology and oncology, and more particularly, the present invention relates to a new target for diagnosis and treatment of chemotherapy-resistant small cell lung cancer and its application.
背景技术Background technique
肺癌是高发病率和高死亡率的恶性肿瘤，根据世界卫生组织统计，肺癌是当今在全球造成死亡人数第一的癌症。近50年来许多国家都报道肺癌的发病率和死亡率均明显增高。而在中国，由于大量的吸烟人群和环境的不断恶化，肺癌已经成为中国发病率最高的一类癌症。诸多因素阻碍了肺癌治疗方案的改进和完善程，目前五年存活率尚不足10％。Lung cancer is a malignant tumor with high morbidity and mortality. According to the statistics of the World Health Organization, lung cancer is the leading cancer causing death in the world today. In the past 50 years, many countries have reported that the incidence and mortality of lung cancer have increased significantly. In China, due to a large number of smokers and the continuous deterioration of the environment, lung cancer has become the highest incidence of cancer in China. Many factors hinder the improvement and perfection of lung cancer treatment programs, and the current five-year survival rate is still less than 10%.
肺癌有多种亚型，根据其组织形态差异，可分为非小细胞肺癌(NSCLC；占肺癌的80-85％)和小细胞肺癌(SCLC；占肺癌的15-20％)。非小细胞肺癌可细分为肺腺癌，肺鳞癌和大细胞肺癌。小细胞肺癌由于其高转移和治疗手段单一的局限，是肺癌中恶性程度最高、预后最差的亚型。小细胞肺癌多发生于肺中央部，生长迅速、转移较早。在对于小细胞肺癌治疗上，因确诊时大部分患者已经发生癌细胞转移，手术适应症窄，大概仅2-5％的患者适合手术治疗。依托泊苷(VP-16)联合顺铂(DDP)方案，因具有化疗效果好而且毒性较轻成为一线治疗的标准方案。化疗可以缓解小细胞肺癌患者症状和延长患者的生存期，但耐药很快发生，使的目前小细胞肺癌患者的生存率不理想，5年生存率低于5％。过去30年中，由于小细胞肺癌患者的肿瘤样本难获取，导致对其发病机制的认知严重滞后。小细胞肺癌患者的生存期和治疗方法基本没有发生较大变化。There are various subtypes of lung cancer, which can be divided into non-small cell lung cancer (NSCLC; 80-85% of lung cancers) and small cell lung cancer (SCLC; 15-20% of lung cancers) according to their histological differences. Non-small cell lung cancer can be subdivided into lung adenocarcinoma, lung squamous cell carcinoma and large cell lung cancer. Small cell lung cancer is the subtype of lung cancer with the highest degree of malignancy and the worst prognosis due to its high metastasis and the limitation of single treatment. Small cell lung cancer mostly occurs in the central part of the lung, grows rapidly and metastasizes earlier. In the treatment of small cell lung cancer, because most patients have already metastasized at the time of diagnosis, the surgical indications are narrow, and only about 2-5% of patients are suitable for surgical treatment. Etoposide (VP-16) combined with cisplatin (DDP) has become the standard first-line treatment because of its good chemotherapy effect and less toxicity. Chemotherapy can relieve the symptoms and prolong the survival period of patients with small cell lung cancer, but drug resistance occurs quickly, so the current survival rate of small cell lung cancer patients is not ideal, and the 5-year survival rate is less than 5%. Over the past 30 years, the lack of access to tumor samples from patients with small cell lung cancer has led to a serious lag in understanding its pathogenesis. The survival and treatment methods of patients with small cell lung cancer have not changed much.
细胞拥有复杂而多样的代谢途径来支撑自身的生长和增殖，肿瘤细胞尤其依赖于多样的代谢途径，来合成其快速增殖所需的物质和能量。鉴于肿瘤细胞过度依赖于重编程后的代谢途径来支撑它们的生存和增殖，可以针对肿瘤细胞的代谢特异性来开展靶向治疗。所以深入研究肿瘤代谢和其生存增殖的关系尤显重要。Cells have complex and diverse metabolic pathways to support their own growth and proliferation. Tumor cells in particular rely on diverse metabolic pathways to synthesize substances and energy required for their rapid proliferation. Given that tumor cells are overly reliant on reprogrammed metabolic pathways to support their survival and proliferation, targeted therapy can target the metabolic specificity of tumor cells. Therefore, it is particularly important to study the relationship between tumor metabolism and its survival and proliferation.
从上世纪七十年代开始，化疗特别是依托泊苷联合顺铂(E/P方案)已经成为临床上治疗小细胞肺癌的首选方案。尽管化疗一开始疗效显著，约80％局限期患者和几乎全部的广泛期患者在治疗一年内出现肿瘤顽固性耐药和恶性进展，由于耐药机制的认识匮乏导致临床上面临对耐药患者基本无药可用。基于伦理限制，几乎不可能通过让这些耐药患者接受活检获得标本来研究小细胞肺癌耐药的机制。而这更进一步导致人们对耐药机制的认识匮乏。因此，在小细胞肺癌临床治疗过程中存在着这样一个怪圈：化疗一线治疗的高效性导致手术治疗不能普及，从而使得临床样本获取非常困难，而 这直接导致人们对化疗耐药机制认识的匮乏，这种认识的匮乏直接影响了新的治疗策略和方案的产生，致使患者耐药后面临基本无药可治的困境。在伦理限制下，临床耐药样本的获取更加困难，进一步导致人们对小细胞肺癌耐药机制的认识不够，最终进入一个难以打开的死结。这个小细胞肺癌耐药研究的死结从上世纪七十年代一直延续至今，在将近五十年的时间里，患者一旦发生耐药几乎无一例外地面临无药可治的处境。Since the 1970s, chemotherapy, especially etoposide combined with cisplatin (E/P regimen), has become the first choice for the clinical treatment of small cell lung cancer. Although chemotherapy is effective at the beginning, about 80% of patients with limited-stage disease and almost all patients with extensive-stage disease develop intractable tumor resistance and malignant progression within one year of treatment. No medicine is available. Due to ethical constraints, it is nearly impossible to study the mechanisms of drug resistance in SCLC by subjecting these drug-resistant patients to biopsy-derived specimens. This further contributes to the lack of understanding of the mechanisms of resistance. Therefore, there is such a vicious circle in the clinical treatment of small cell lung cancer: the high efficiency of first-line chemotherapy leads to the unpopularity of surgical treatment, which makes it very difficult to obtain clinical samples, which directly leads to the lack of understanding of the mechanism of chemotherapy resistance. This lack of understanding directly affects the generation of new treatment strategies and programs, resulting in patients facing the dilemma of basically no cure after drug resistance. Under ethical constraints, it is more difficult to obtain clinical drug resistance samples, which further leads to insufficient understanding of the drug resistance mechanism of small cell lung cancer, and finally enters a dead end that is difficult to open. The dead end of drug resistance research in small cell lung cancer has continued since the 1970s. For nearly 50 years, patients who develop drug resistance are almost invariably faced with no cure.
因此，建立有效的研究体系进行小细胞肺癌化疗耐药的机制研究迫在眉睫；同时，本领域也亟待开发新的小细胞肺癌治疗药物。Therefore, it is urgent to establish an effective research system to study the mechanism of chemoresistance in small cell lung cancer; at the same time, there is also an urgent need to develop new small cell lung cancer drugs in this field.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供诊断和治疗化疗耐药小细胞肺癌的新靶点及其应用。本发明涉及一类化疗耐药小细胞肺癌治疗新靶点的发现与应用。公开了小细胞肺癌化疗耐药小鼠模型的构建方法；还公开了小分子抑制剂他汀类药物是通过香叶基香叶基焦磷酸(GGPP)-RAB7A-自噬途径选择性抑制化疗耐药小细胞癌的生长；还公开了敲低GGPP合成酶(GGPS1)或RAB7A的基因表达可以选择性抑制化疗耐药小细胞癌肿瘤的增殖；还公开了他汀类药物及其与化疗药物联用可以选择性抑制化疗耐药小细胞癌，尤其是高表达GGPS1的化疗耐药小细胞癌肿瘤的生长；本发明首次揭示了一类化疗耐药小细胞肺癌的新治疗靶点及其应用，为针对小细胞肺癌化疗耐药的治疗提供了全新的思路和方法。The purpose of the present invention is to provide a new target for diagnosing and treating chemotherapy-resistant small cell lung cancer and its application. The present invention relates to the discovery and application of a new therapeutic target for chemotherapy-resistant small cell lung cancer. Disclosed is a method for constructing a chemoresistance mouse model of small cell lung cancer; it is also disclosed that small molecule inhibitor statins selectively inhibit chemoresistance through geranylgeranyl pyrophosphate (GGPP)-RAB7A-autophagy pathway Growth of small cell carcinoma; also disclosed that knocking down the gene expression of GGPP synthase (GGPS1) or RAB7A can selectively inhibit the proliferation of chemotherapy-resistant small cell carcinoma tumors; also disclosed that statins and their combination with chemotherapeutic drugs can Selectively inhibits the growth of chemotherapy-resistant small cell carcinoma, especially chemotherapy-resistant small cell carcinoma with high expression of GGPS1; the present invention discloses for the first time a new therapeutic target of a class of chemotherapy-resistant small cell lung cancer and its application, which is aimed at targeting The treatment of chemotherapy resistance in small cell lung cancer provides a new idea and method.
在本发明的第一方面，提供一种筛选抑制化疗耐药小细胞肺癌的物质的方法，所述方法包括：(1)将候选物质与含有GGPS1(GGPP合成酶)/RAB7A(Ras相关蛋白7A)/自噬流信号通路的体系接触；(2)筛选出调节GGPS1/RAB7A/自噬流信号通路的物质，所述物质是对于抑制化疗耐药小细胞肺癌有用的物质(包括潜在物质)；其中，所述的调节包括：抑制GGPS1的表达或活性，抑制RAB7A的膜定位，抑制GGPS1代谢产物GGPP对RAB7A的修饰作用，或促进自噬流障碍。In a first aspect of the present invention, there is provided a method for screening a substance for inhibiting chemotherapy-resistant small cell lung cancer, the method comprising: (1) combining a candidate substance with GGPS1 (GGPP synthase)/RAB7A (Ras-related protein 7A) )/autophagy flux signaling pathway system contact; (2) screen out substances that regulate GGPS1/RAB7A/autophagy flux signaling pathway, and the substances are useful substances (including potential substances) for inhibiting chemotherapy-resistant small cell lung cancer; The regulation includes: inhibiting the expression or activity of GGPS1, inhibiting the membrane localization of RAB7A, inhibiting the modification effect of GGPS1 metabolite GGPP on RAB7A, or promoting autophagic flow disorder.
在一个优选例中，所述的GGPS1/RAB7A/自噬流信号通路被包含于甲羟戊酸途径中，或为甲羟戊酸途径的下游通路。In a preferred embodiment, the GGPS1/RAB7A/autophagy flux signaling pathway is included in the mevalonate pathway, or is a downstream pathway of the mevalonate pathway.
在另一优选例中，所述的GGPS1/RAB7A/自噬流信号通路包括：GGPS1蛋白，RAB7A蛋白；所述自噬流为自噬体与溶酶体相互融合引发的自噬流。In another preferred embodiment, the GGPS1/RAB7A/autophagic flux signaling pathway includes: GGPS1 protein, RAB7A protein; the autophagic flux is autophagic flux triggered by the fusion of autophagosome and lysosome.
在另一优选例中，步骤(1)包括：向含有GGPS1/RAB7A/自噬流信号通路的体系中添加候选物质；步骤(2)包括：检测GGPS1/RAB7A/自噬流信号通路中各蛋白或其编码基因的变化，并与对照组比较，其中所述的对照组是不添加所述候选物质的、含有GGPS1/RAB7A/自噬流信号通路的体系；若候选物质抑制GGPS1的表达或活性，抑制 RAB7A的膜定位，抑制GGPS1代谢产物GGPP对RAB7A的修饰作用，或促进自噬流障碍，则该候选物质是对于抑制化疗耐药小细胞肺癌有用的物质。In another preferred example, step (1) includes: adding candidate substances to the system containing the GGPS1/RAB7A/autophagy flow signaling pathway; step (2) includes: detecting each protein in the GGPS1/RAB7A/autophagy flow signaling pathway Changes in the gene or its encoding gene, and compared with the control group, wherein the control group is a system containing the GGPS1/RAB7A/autophagy flow signaling pathway without adding the candidate substance; if the candidate substance inhibits the expression or activity of GGPS1 , inhibiting the membrane localization of RAB7A, inhibiting the modification of RAB7A by GGPS1 metabolite GGPP, or promoting autophagy flow disorder, the candidate substance is a useful substance for inhibiting chemotherapy-resistant small cell lung cancer.
在另一优选例中，所述的含有GGPS1/RAB7A/自噬流信号通路的体系选自：细胞(培养物)体系、亚细胞(培养物)体系、组织(培养物)体系或动物体系。In another preferred embodiment, the system containing the GGPS1/RAB7A/autophagy flow signaling pathway is selected from: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system or an animal system.
在另一优选例中，所述的提高或促进为统计学上的提高或促进，如与对照或基底相比，提高或促进10％或20％以上，较佳地提高或促进40％或50％以上，更佳地提高或促进80％或100％以上。In another preferred embodiment, the improvement or promotion is a statistical improvement or promotion, for example, compared with the control or the base, the improvement or promotion is more than 10% or 20%, preferably 40% or 50%. % or more, more preferably 80% or 100% or more.
在另一优选例中，所述的抑制也可被称为下调，为统计学上的抑制或下调，如与对照或基底相比，抑制或下调10％或20％以上，较佳地抑制或下调40％或50％以上，更佳地抑制或下调80％或100％以上。In another preferred embodiment, the inhibition can also be referred to as down-regulation, which is a statistical inhibition or down-regulation, such as inhibiting or down-regulation by more than 10% or 20% compared with the control or the base, preferably inhibiting or down-regulation. Down 40% or 50% or more, more preferably 80% or 100% or more.
在另一优选例中，所述的候选物质包括(但不限于)：针对GGPS1/RAB7A/自噬流信号通路、或其通路蛋白、或其上游或下游蛋白或基因设计的调控分子(如但不限于上调剂、干扰分子、核酸抑制物、结合分子(如抗体或配体))，CRISPR构建物，小分子化合物，来自化合物库的化合物。In another preferred example, the candidate substances include (but are not limited to): regulatory molecules (such as but Without limitation, upregulators, interfering molecules, nucleic acid inhibitors, binding molecules (eg, antibodies or ligands), CRISPR constructs, small molecule compounds, compounds from compound libraries.
在本发明的另一方面，提供GGPS1/RAB7A/自噬流信号通路的用途，用于筛选抑制化疗耐药小细胞肺癌的物质；较佳地，所述的GGPS1/RAB7A/自噬流信号通路被包含于甲羟戊酸途径中，或为甲羟戊酸途径的下游通路；或，所述的GGPS1/RAB7A/自噬流信号通路包括：GGPS1蛋白，RAB7A蛋白；所述自噬流为自噬体与溶酶体相互融合引发的自噬流。In another aspect of the present invention, the use of the GGPS1/RAB7A/autophagy flow signaling pathway is provided for screening substances that inhibit chemotherapy-resistant small cell lung cancer; preferably, the GGPS1/RAB7A/autophagic flow signaling pathway It is included in the mevalonate pathway, or is a downstream pathway of the mevalonate pathway; or, the GGPS1/RAB7A/autophagy flow signaling pathway includes: GGPS1 protein, RAB7A protein; the autophagy flow is autophagy. Autophagic flux triggered by the fusion of phagosomes and lysosomes.
在本发明的另一方面，提供调节GGPS1/RAB7A/自噬流信号通路的调节剂的用途，用于制备抑制化疗耐药小细胞肺癌的药物组合物；其中，所述的调节剂包括选自下组：GGPS1的表达或活性抑制剂，RAB7A膜定位抑制剂，GGPS1代谢产物GGPP对RAB7A的修饰作用的抑制剂，或促进自噬流障碍的促进剂。In another aspect of the present invention, there is provided the use of a regulator that modulates the GGPS1/RAB7A/autophagy flow signaling pathway, for the preparation of a pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer; wherein, the regulator comprises a compound selected from the group consisting of Lower panel: inhibitors of GGPS1 expression or activity, inhibitors of RAB7A membrane localization, inhibitors of the modification of RAB7A by GGPS1 metabolite GGPP, or promoters that promote disorders of autophagic flux.
在一个优选例中，所述GGPS1的表达或活性抑制剂包括(但不限于)：敲除或沉默GGPS1基因的试剂，抑制GGPS1蛋白活性的试剂；较佳地，包括：特异性干扰GGPS1基因表达的干扰分子，针对GGPS1基因的CRISPR基因编辑试剂、同源重组试剂或定点突变试剂，所述试剂将GGPS1进行功能丧失性突变。In a preferred embodiment, the GGPS1 expression or activity inhibitor includes (but is not limited to): an agent for knocking out or silencing GGPS1 gene, an agent for inhibiting GGPS1 protein activity; preferably, including: specifically interfering with GGPS1 gene expression A CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent targeting the GGPS1 gene, the reagent mutates GGPS1 with loss-of-function.
在另一优选例中，所述RAB7A膜定位抑制剂包括(但不限于)：敲除或沉默RAB7A基因的试剂，抑制RAB7A蛋白活性的试剂；较佳地，包括：特异性干扰RAB7A基因表达的干扰分子，针对RAB7A基因的CRISPR基因编辑试剂、同源重组试剂或定点突变试剂，所述试剂将RAB7A进行功能丧失性突变。In another preferred embodiment, the RAB7A membrane localization inhibitor includes (but is not limited to): an agent that knocks out or silences the RAB7A gene, and an agent that inhibits the activity of the RAB7A protein; preferably, it includes: specifically interferes with the expression of the RAB7A gene Interfering molecules, CRISPR gene editing reagents, homologous recombination reagents, or site-directed mutagenesis reagents targeting the RAB7A gene that mutate RAB7A loss-of-function.
在另一优选例中，所述GGPS1代谢产物GGPP对RAB7A的修饰作用的抑制剂包括(但不限于)：抑制GGPS1合成GGPP的试剂。In another preferred example, the inhibitor of the modification effect of the GGPS1 metabolite GGPP on RAB7A includes (but is not limited to): an agent that inhibits the synthesis of GGPP by GGPS1.
在另一优选例中，所述促进自噬流障碍的促进剂包括(但不限于)：GGPS1的表达或活性抑制剂，RAB7A膜定位抑制剂，GGPS1代谢产物GGPP对RAB7A的修饰作用的抑制剂。In another preferred embodiment, the promoters promoting autophagic flow disorder include (but are not limited to): inhibitors of GGPS1 expression or activity, inhibitors of RAB7A membrane localization, inhibitors of modification of RAB7A by GGPS1 metabolite GGPP .
在另一优选例中，所述的敲除或沉默GGPS1基因的试剂为敲低GGPS1的shRNA质粒；用于敲低的功能序列为： CCTGAGCTAGTAGCCTTAGTA所述的敲除或沉默RAB7A基因的试剂为敲低RAB7A的shRNA质粒；用于敲低的功能序列为：GGCTAGTCACAATGCAGATAT。 In another preferred example, the reagent for knocking out or silencing the GGPS1 gene is a shRNA plasmid for knocking down GGPS1; the functional sequence for knocking down is: C CTGAGCTAGTAGCCTTAGTA The reagent for knocking out or silencing the RAB7A gene is knock-down Low RAB7A shRNA plasmid; the functional sequence for knockdown is: GGCTAGTCACAATGCAGATAT.
在本发明的另一方面，提供他汀类药物、依托泊苷和顺铂的组合在制备抑制化疗耐药小细胞肺癌的药物组合物中的用途。In another aspect of the present invention, there is provided the use of a combination of statins, etoposide and cisplatin in the preparation of a pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer.
在本发明的另一方面，提供一种抑制化疗耐药小细胞肺癌的药物组合物，包括他汀类药物、依托泊苷和顺铂。In another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer, comprising statins, etoposide and cisplatin.
在本发明的另一方面，提供一种抑制化疗耐药小细胞肺癌的药盒，其中包括所述的药物组合物；或其中包括容器，以及分别置于所述容器中的他汀类药物、依托泊苷和顺铂。In another aspect of the present invention, there is provided a kit for inhibiting chemotherapy-resistant small cell lung cancer, which includes the pharmaceutical composition; or a container, and the statins, etiology, etc. respectively placed in the container. Poside and Cisplatin.
在一个优选例中，所述他汀类药物包括选自：美伐他汀(MevaStatin)，辛伐他汀(SimvaStatin)，匹伐他汀(PitvaStatin)，阿托伐他汀(AtorvStatin)，洛伐他汀(LovaStatin)，FluaStatin。In a preferred example, the statins include Mevastatin, Simvastatin, PitvaStatin, Atorvastatin, LovaStatin. , FluaStatin.
在另一优选例中，所述的化疗耐药小细胞肺癌为高表达GGPS1耐药性的小细胞肺癌。In another preferred embodiment, the chemotherapy-resistant small cell lung cancer is a small cell lung cancer with high expression of GGPS1 resistance.
在另一优选例中，所述的“高表达”为统计学意义上的“高表达”，例如，“高表达GGPS1的小细胞肺癌”与“小细胞肺癌”总人群(或统计学意义上足够量的人群)的平均GGPS1的表达相比，显著高10％或20％以上，较佳地高30％或50％以上，更佳地高80％或100％以上。In another preferred embodiment, the "high expression" is "high expression" in a statistical sense, for example, "small cell lung cancer with high expression of GGPS1" and the total population of "small cell lung cancer" (or statistical significance) A sufficient amount of the population) is significantly higher than the average GGPS1 expression of 10% or 20%, preferably 30% or 50% higher, more preferably 80% or 100% higher.
在另一优选例中，用于一个单位疗程时，所述的他汀类药物、依托泊苷和顺铂的用量比例为(35～105)：(3～9)：1；较佳地为(49～70)：(4.5～7.5)：1(如58.3：5：1)。In another preferred example, when used for one unit course of treatment, the dosage ratio of the statins, etoposide and cisplatin is (35-105):(3-9):1; preferably ( 49 to 70): (4.5 to 7.5): 1 (eg 58.3: 5: 1).
在另一优选例中，所述顺铂、依托泊苷和所述他汀类药物的用药方法为：按1周1个疗程计算，根据个体体重，通过腹腔注射方式第1天给予0.5～10毫克/千克(如0.8～6毫克/千克，更具体如1、2、3、4、5毫克/千克)顺铂(CDDP)；第1～3天给予0.5～15毫克/千克/天(如1～12毫克/千克/天，更具体如2、4、6、8、10毫克/千克/天)依托泊苷(VP16)；同时通过灌胃方式给予2～100毫克/千克/天(如5～60毫克/千克/天，更具体如6、8、10、15、20、30、40、50毫克/千克/天)的美伐他汀或辛伐他汀或匹伐他汀等药物处理；较佳地，当药物被置于药盒中时，所述用药方法被记载于所述药盒的使用说明书中。In another preferred example, the method of administration of the cisplatin, etoposide and the statin is as follows: 0.5-10 mg is administered on the first day by intraperitoneal injection according to the body weight of the individual, calculated as one course of treatment per week. /kg (eg 0.8-6 mg/kg, more specifically such as 1, 2, 3, 4, 5 mg/kg) cisplatin (CDDP); 0.5-15 mg/kg/day (eg 1 to 3 days) ~12 mg/kg/day, more specifically 2, 4, 6, 8, 10 mg/kg/day) etoposide (VP16); concurrently 2 to 100 mg/kg/day (eg 5 mg/kg/day) by gavage ~60 mg/kg/day, more specifically such as 6, 8, 10, 15, 20, 30, 40, 50 mg/kg/day) of mevastatin or simvastatin or pitavastatin and other drug treatments; preferably Typically, when the drug is placed in a kit, the method of administration is described in the instructions for use of the kit.
在本发明的另一方面，提供GGPS1蛋白或其编码基因在制备诊断试剂中的用途，所述的诊断试剂用于对小细胞肺癌进行诊断或预后；较佳地，所述的诊断或预后包括：根据GGPS1蛋白的表达情况，判断其是否适用于进行他汀类药物的治疗方案；若GGPS1蛋白高表达，则适用该治疗方案。In another aspect of the present invention, there is provided the use of GGPS1 protein or its encoding gene in preparing a diagnostic reagent for diagnosing or prognosing small cell lung cancer; preferably, the diagnosing or prognosticating comprises: : According to the expression of GGPS1 protein, determine whether it is suitable for statin therapy; if GGPS1 protein is highly expressed, this therapy is applicable.
在本发明的另一方面，提供特异性识别GGPS1蛋白或其编码基因的试剂的用途，用于制备对小细胞肺癌进行诊断或预后的诊断试剂或诊断试剂盒；较佳地，所述的诊断或预后包括：根据GGPS1蛋白的表达情况，判断其是否适用于进行他汀类药物的治疗方案；若GGPS1蛋白高表达，则适用该治疗方案。In another aspect of the present invention, there is provided the use of a reagent that specifically recognizes GGPS1 protein or its encoding gene, for preparing a diagnostic reagent or a diagnostic kit for diagnosing or prognosing small cell lung cancer; Or prognosis includes: according to the expression of GGPS1 protein, judging whether it is suitable for the treatment plan of statin; if the GGPS1 protein is highly expressed, the treatment plan is applicable.
在一个优选例中，所述的治疗方案为他汀类药物、依托泊苷和顺铂联合用药的治疗方案。In a preferred example, the treatment regimen is a treatment regimen of a combination of statins, etoposide and cisplatin.
在另一优选例中，所述的诊断试剂包括选自：特异性扩增GGPS1蛋白的编码基因的引物；特异性识别GGPS1蛋白的编码基因或其转录本的探针；或特异性抗GGPS1蛋白的抗体。In another preferred embodiment, the diagnostic reagent comprises a primer selected from the group consisting of: a primer that specifically amplifies the gene encoding GGPS1 protein; a probe that specifically recognizes the gene encoding GGPS1 protein or its transcript; or a specific anti-GGPS1 protein of antibodies.
在本发明的另一方面，提供一种用于对小细胞肺癌进行诊断或预后的试剂盒，所述的试剂盒中含有：检测GGPS1蛋白或其编码基因的表达情况或表达量的诊断试剂。In another aspect of the present invention, a kit for diagnosing or prognosing small cell lung cancer is provided, wherein the kit contains: a diagnostic reagent for detecting the expression or expression level of GGPS1 protein or its encoding gene.
在另一优选例中，所述的试剂盒中还包括：核酸抽提试剂，聚合酶链反应试剂，蛋白免疫印迹试剂，和/或酶链免疫反应试剂。In another preferred embodiment, the kit further includes: nucleic acid extraction reagents, polymerase chain reaction reagents, western blotting reagents, and/or enzyme chain immunoreaction reagents.
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.
附图说明Description of drawings
图1、建立小鼠小细胞肺癌化疗耐药肿瘤的方案。Figure 1. Scheme for establishing chemotherapy-resistant tumors in mouse small cell lung cancer.
A.模型建立和实验流程图。皮下成瘤的动物会每周给予依托泊苷/顺铂(E/P)或对照的处理，通过不断的肿瘤传代接种直至产生化疗耐药模型。A. Model establishment and experimental flow chart. Subcutaneously tumorigenic animals will be treated with etoposide/cisplatin (E/P) or control weekly and inoculated by successive tumor passages until a chemoresistance model is developed.
B.根据临床上的标准流程给药。按照1周1个疗程计算，第1天：顺铂处理，第1-3天：依托泊苷处理，第4-7天，不给药。B. Dosing according to standard clinical procedures. According to the calculation of one course of treatment per week, the first day: cisplatin treatment, the first-3 days: etoposide treatment, the 4-7 days, no administration.
图2、建立小鼠模型的小细胞肺癌化疗耐药肿瘤。Figure 2. A mouse model of small cell lung cancer chemotherapy-resistant tumors.
A.每一代敏感细胞系体内对于E/P药物的效应。对照组(绿色)和化疗处理组(红色)数据以单线条的显示呈现。A. The effect of each generation of sensitive cell lines on E/P drugs in vivo. Control (green) and chemotherapy-treated (red) data are presented as single-line displays.
B.对敏感和耐药肿瘤在给与E/P药物情况下生存分析，采用log rank(Mantle-Cox)检验。本发明人定义肿瘤体积达到1000立方毫米时为小鼠的生存终点。B. Survival analysis of sensitive and resistant tumors under E/P drug administration, using log rank (Mantle-Cox) test. The inventors defined the survival endpoint of mice when the tumor volume reached 1000 cubic millimeters.
C.比较肿瘤H82，H209，H526，H146和H82R，H209R，H526R，H146R之间E/P的抑制效率和小鼠生存周期。C. Comparison of E/P inhibition efficiency and mouse survival time between tumors H82, H209, H526, H146 and H82R, H209R, H526R, H146R.
D.H82和H82R肿瘤在给予E/P处理情况下的，凋亡相关标志物Cleaved-Caspase 3(CC3)的免疫组化染色。比例尺：50μm。后为H82和H82R肿瘤在给予E/P处理情况下的CC3染色统计图。D. Immunohistochemical staining of apoptosis-related marker Cleaved-Caspase 3 (CC3) in H82 and H82R tumors treated with E/P. Scale bar: 50 μm. The following is the CC3 staining statistics of H82 and H82R tumors treated with E/P.
E.小细胞化疗耐药细胞系(H446，DMS114)体内每一代肿瘤对于E/P的响应。对照组(绿色)和化疗处理组(红色)数据以单线条的显示呈现。E. Small cell chemoresistant cell lines (H446, DMS114) in vivo tumor response to E/P at each generation. Control (green) and chemotherapy-treated (red) data are presented as single-line displays.
F.对耐药肿瘤在给与E/P药物情况下生存分析，用log rank(Mantle-Cox)检验。F. Survival analysis of drug-resistant tumors under E/P drug administration, using log rank (Mantle-Cox) test.
G.在耐药肿瘤H446和DMS114，检测E/P的抑制效率和小鼠生存周期。G. In drug-resistant tumors H446 and DMS114, the inhibition efficiency of E/P and the mouse survival cycle were examined.
所有数据分析采用双因素方差分析或者未配对的t检验。生存分析采用log-rank(Mantel–Cox)检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. Survival analysis was performed using the log-rank (Mantel–Cox) test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图3、建立小细胞肺癌化疗耐药细胞株。Figure 3. Establishment of chemotherapy-resistant cell lines for small cell lung cancer.
A.在E/P处理下，通过检测细胞活力检测H82和H82R细胞的IC50。A. IC50 of H82 and H82R cells was detected by detecting cell viability under E/P treatment.
B.体外检测H82和H82R细胞对于E/P处理的响应。B. Response of H82 and H82R cells to E/P treatment in vitro.
C.体外检测H526和H526R细胞对于E/P处理的响应。C. In vitro detection of the response of H526 and H526R cells to E/P treatment.
D.体外检测H209和H209R细胞对于E/P处理的响应。D. Response of H209 and H209R cells to E/P treatment in vitro.
E.体外检测化疗耐药细胞H446，DMS114和H196对于E/P处理的响应。E. Response of chemoresistant cells H446, DMS114 and H196 to E/P treatment in vitro.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图4、用FDA认证的代谢类药物进行药库筛选。Figure 4. Drug library screening with FDA-approved metabolites.
A.利用H82和H82R细胞进行药库筛选的流程。有256个小分子抑制剂被用于药库筛选。每孔按2000细胞铺下，24小时后按5微摩尔的浓度加入药库药物，在第四天检测细胞活力。A. The flow of drug library screening using H82 and H82R cells. There are 256 small molecule inhibitors used for drug library screening. Each well was plated with 2000 cells, and after 24 hours, the drug library was added at a concentration of 5 micromolar, and the cell viability was detected on the fourth day.
B.对于药库按照应用的人类疾病分类。B. Human disease classification for drug libraries by application.
C.对于药库按照所属的代谢通路分类。C. For drug libraries, they are classified according to the metabolic pathway they belong to.
图5、他汀类药物抑制H82R细胞生长。Figure 5. Statins inhibit H82R cell growth.
A.比较H82R/H82的生长差异，得出药物的筛选抑制效率(每个药物三次重复)。红色点代表的药物显著抑制H82R细胞生长(log2大于0.5，p值小于0.05)，蓝色点代表的药物显著抑制H82细胞生长(log2小于-0.5，p值小于0.05)。他汀类药物被发现相对于H82细胞，可以显著抑制H82R细胞生长。A. Comparing the growth difference of H82R/H82, the screening inhibition efficiency of the drug was obtained (each drug was repeated three times). Drugs represented by red dots significantly inhibited the growth of H82R cells (log2 greater than 0.5, p value less than 0.05), and drugs represented by blue dots significantly inhibited H82 cell growth (log2 less than -0.5, p value less than 0.05). Statins were found to significantly inhibit H82R cell growth relative to H82 cells.
B.在细胞给予MevaStatin按照2μM、5μM和10μM处理72小时，检测H82和H82R细胞生长。B. H82 and H82R cell growth was detected after cells were treated with MevaStatin at 2 μM, 5 μM and 10 μM for 72 hours.
C.在细胞给予不同浓度他汀(AtorvStatin、PitvaStatin、LovaStatin、MevaStatin、SimvaStatin和FluaStatin)处理后，检测H82和H82R细胞的生长。D.在细胞给予对照处理，E/P处理，美伐他汀(MevaStatin，Meva)处理和联合处理后，连续四天每天检测H82和H82R细胞生长。C. The growth of H82 and H82R cells was examined after cells were treated with different concentrations of statins (AtorvStatin, PitvaStatin, LovaStatin, MevaStatin, SimvaStatin and FluaStatin). D. H82 and H82R cell growth was measured daily for four consecutive days after cells were given control treatment, E/P treatment, mevastatin (MevaStatin, Meva) treatment and combination treatment.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图6、他汀类药物抑制多株小细胞肺癌化疗耐药细胞的生长。Figure 6. Statins inhibit the growth of chemotherapy-resistant cells in multiple small cell lung cancer strains.
A.化疗敏感细胞系(H146、H209和H526)和化疗耐药细胞系(H196、H446和DMS114)在给予对照处理，E/P处理，MevaStatin处理和联合处理的情况下，连续四天每天检测细胞的增殖。A. Chemosensitive cell lines (H146, H209 and H526) and chemoresistant cell lines (H196, H446 and DMS114) were tested daily for four consecutive days in the presence of control, E/P, MevaStatin and combination treatments proliferation of cells.
B.比较化疗敏感细胞系和化疗耐药系对于E/P的响应。比较化疗敏感细胞系和化疗耐药细胞系对于5μM MevaStatin的响应。处理72小时后，WB检测蛋白PARP、CC3、P21和P27水平。B. Comparison of the responses of chemosensitive and chemoresistant cell lines to E/P. The response of chemosensitive and chemoresistant cell lines to 5 μM MevaStatin was compared. After 72 hours of treatment, protein levels of PARP, CC3, P21 and P27 were detected by WB.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图7、他汀类药物抑制H82R移植瘤生长。Figure 7. Statins inhibit the growth of H82R xenografts.
A.H82R细胞皮下成瘤后，分成四组：按照对照处理，E/P处理，MevaStatin处理和联A. H82R cells were divided into four groups after subcutaneous tumor formation: control treatment, E/P treatment, MevaStatin treatment and combination treatment
合处理。肿瘤体积用游标卡尺测量后计算。图中显示了每组肿瘤增长曲线图(左)，combined processing. Tumor volume was calculated after measuring with vernier calipers. The graphs show tumor growth curves for each group (left),
每组肿瘤重量图(中)，每组小鼠体重图(右)。Tumor weight graph for each group (middle), and mouse weight graph for each group (right).
B.H82R肿瘤在不同药物处理后，对于每组的肿瘤取后拍照。B. After H82R tumor was treated with different drugs, the tumor of each group was taken and photographed.
C.免疫组化显示每组药物处理后，H82R肿瘤凋亡标志物CC3和DNA损伤标志物C. Immunohistochemistry showed that H82R tumor apoptosis marker CC3 and DNA damage marker after each drug treatment
H2AX染色。比例尺：50μm。H2AX staining. Scale bar: 50 μm.
D.对于H82R每组肿瘤CC3和H2AX的染色比例统计图。E.H82R细胞皮下成瘤后，分成多组按照对照处理，E/P处理，SimvaStatin处理，PitvaStatin处理和联合处理。图中显示了每组肿瘤增长曲线图(左)，每组肿瘤重量图(中)，每组小鼠体重图(右)。D. Statistical graph of the staining ratio of CC3 and H2AX in each group of tumors for H82R. After subcutaneous tumor formation, E.H82R cells were divided into multiple groups according to control treatment, E/P treatment, SimvaStatin treatment, PitvaStatin treatment and combined treatment. The graphs show a graph of tumor growth in each group (left), a graph of tumor weight in each group (middle), and a graph of mouse body weight in each group (right).
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图8、GGPP逆转Statin对H82R细胞的抑制效应。Figure 8. GGPP reverses the inhibitory effect of Statin on H82R cells.
A.甲羟戊酸(MVA)、香叶基香叶基焦磷酸(GGPP)和香叶基香叶醇(GGOH)的加入可以回复5μM MevaStatin处理对于H82R细胞的抑制效应，但加入法尼焦磷酸(FPP)、法尼醇(FFOH)、鲨烯(SQ)、辅酶Q9(CoQ9)和辅酶Q10(CoQ10)无法回复5μM MevaStatin处理对于H82R细胞的抑制效应。A. The addition of mevalonic acid (MVA), geranylgeranyl pyrophosphate (GGPP) and geranylgeraniol (GGOH) can restore the inhibitory effect of 5 μM MevaStatin treatment on H82R cells, but the addition of farnesyl pyrophosphate (FPP), farnesol (FFOH), squalene (SQ), coenzyme Q9 (CoQ9) and coenzyme Q10 (CoQ10) could not restore the inhibitory effect of 5 μM MevaStatin treatment on H82R cells.
B.检测不同浓度的FPP、FFOH、GGPP、GGOH、SQ或CoQ9、CoQ10加入是否可以回复5μM MevaStatin处理对于H82R细胞的抑制效应。B. Detect whether the addition of different concentrations of FPP, FFOH, GGPP, GGOH, SQ or CoQ9, CoQ10 can restore the inhibitory effect of 5 μM MevaStatin treatment on H82R cells.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图9、Statin通过抑制GGPP产生影响RAB7A的胞内分布来抑制H82R细胞。Figure 9. Statin inhibits H82R cells by inhibiting GGPP production and affecting the intracellular distribution of RAB7A.
A.RNA-seq检测大部分RAB蛋白家族基因的FKM值。A. RNA-seq detection of FKM values of most RAB protein family genes.
B.热图分析由小G蛋白和MVA通路的基因构成的shRNA库对于H82R细胞生长影响。B. Heat map analysis of the effect of shRNA pools composed of genes of small G protein and MVA pathway on the growth of H82R cells.
C.利用Tet-on系统，四环霉素(Doxycycline)处理H82和H82R细胞会诱导GGPS1和RAB7A的敲低。C. Doxycycline treatment of H82 and H82R cells induced knockdown of GGPS1 and RAB7A using the Tet-on system.
D.细胞在对照处理、5μM MevaStatin、5μM MevaStatin和2μM GGPP处理72小时后，检测RAB7A在细胞的胞质内，膜上和总的表达水平。钠/钾离子转运ATP酶α1肽(ATP1A1)为特异性表达在细胞膜上的蛋白，微管蛋白(Tubulin)是内参蛋白。所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。D. Cells were treated with control, 5 μM MevaStatin, 5 μM MevaStatin, and 2 μM GGPP for 72 hours, and the expression levels of RAB7A in the cytoplasm, membrane and total of cells were detected. The sodium/potassium ion transporting ATPase α1 peptide (ATP1A1) is a protein specifically expressed on the cell membrane, and Tubulin is an internal reference protein. All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图10、Statin通过GGPP-RAB7A-自噬途径引起H82R细胞的自噬障碍。Figure 10. Statin causes autophagy disorder in H82R cells through GGPP-RAB7A-autophagy pathway.
A.H82和H82R细胞在对照处理、5μM MevaStatin、5μM MevaStatin和2μM GGPP处理72小时后，WB检测P62和LC3B的表达水平。A. H82 and H82R cells were treated with control, 5 μM MevaStatin, 5 μM MevaStatin and 2 μM GGPP for 72 hours, and the expression levels of P62 and LC3B were detected by WB.
B.H82和H82R细胞在对照处理、5μM MevaStatin、5μM MevaStatin和2μM GGPP处理72小时后，WB检测PARP和CC3的表达水平。B. H82 and H82R cells were treated with control, 5 μM MevaStatin, 5 μM MevaStatin and 2 μM GGPP for 72 hours, and the expression levels of PARP and CC3 were detected by WB.
C.敏感细胞系(H209、H526)和耐药细胞系(H446、DMS114)细胞在对照处理，5μM MevaStatin处理72小时后，WB检测P62和LC3B的表达水平。C. Sensitive cell lines (H209, H526) and resistant cell lines (H446, DMS114) cells were treated with control and 5 μM MevaStatin for 72 hours, and the expression levels of P62 and LC3B were detected by WB.
D.H82R细胞敲低GGPS1或RAB7A后，WB检测P62和LC3B的表达水平。After knockdown of GGPS1 or RAB7A in D.H82R cells, the expression levels of P62 and LC3B were detected by WB.
E.H82R细胞敲低GGPS1或RAB7A后，WB检测PARP和CC3的表达水平。After knockdown of GGPS1 or RAB7A in E.H82R cells, the expression levels of PARP and CC3 were detected by WB.
F.H82R细胞敲低HMGCR后，WB检测HMGCR、CC3、P62和LC3B的表达水平。After knockdown of HMGCR in F.H82R cells, the expression levels of HMGCR, CC3, P62 and LC3B were detected by WB.
G.检测H446细胞在敲低GGPS1或RAB7A后的细胞活力。G. Detection of cell viability of H446 cells after knockdown of GGPS1 or RAB7A.
H.H446细胞敲低GGPS1或RAB7A后，WB检测P62和LC3B的表达水平。After knockdown of GGPS1 or RAB7A in H.H446 cells, the expression levels of P62 and LC3B were detected by WB.
I.WB检测H82R肿瘤对照组，MevaStatin组，联合处理组中的P62和LC3B的表达水平。I. WB detected the expression levels of P62 and LC3B in H82R tumor control group, MevaStatin group and combined treatment group.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图11、免疫荧光示踪小细胞肺癌化疗耐药细胞的自噬流。Figure 11. Immunofluorescence tracing of autophagic flux in chemotherapy-resistant small cell lung cancer cells.
A.稳转细胞株H82R mRFP-GFP-LC3分别给予对照处理、5μM MevaStatin、5μM MevaStatin和2μM GGPP处理72小时后，统计细胞内自噬溶酶体的数目。A. The number of autophagolysosomes in the cells was counted after the stably transfected cell line H82R mRFP-GFP-LC3 was treated with control, 5 μM MevaStatin, 5 μM MevaStatin and 2 μM GGPP for 72 hours.
B.稳转细胞株H446mRFP-GFP-LC3分别给予对照处理、5μM MevaStatin、5μM MevaStatin和2μM GGPP处理72小时后，统计细胞内自噬溶酶体的数目。B. After the stably transfected cell line H446mRFP-GFP-LC3 was treated with control, 5 μM MevaStatin, 5 μM MevaStatin and 2 μM GGPP for 72 hours, the number of autophagolysosomes in the cells was counted.
C.稳转细胞株H446mRFP-GFP-LC3在GGPS1或RAB7A敲低后，统计细胞内自噬溶酶体的数目。C. After GGPS1 or RAB7A knockdown of H446mRFP-GFP-LC3 stably transfected cell line, the number of autophagolysosomes in cells was counted.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01， ***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图12、敲低GGPS1或RAB7A抑制H82R肿瘤的生长。Figure 12. Knockdown of GGPS1 or RAB7A inhibits H82R tumor growth.
A.H82R诱导敲低GGPS1细胞皮下成瘤后，分别给予对照处理(水，每组五只)和doxycycline(1毫克/毫升溶于饮用水中，每组五只)。肿瘤体积通过游标卡尺测量后计算。图中显示了每组肿瘤增长曲线图(左)，每组肿瘤重量图(中)，肿瘤拍照图(右)。A. After H82R-induced knockdown of GGPS1 cells subcutaneously formed tumors, they were given control treatment (water, five animals in each group) and doxycycline (1 mg/ml dissolved in drinking water, five animals in each group). Tumor volume was calculated by vernier caliper measurement. The figure shows the graph of tumor growth in each group (left), the graph of tumor weight in each group (middle), and the graph of tumor photography (right).
B.H82R肿瘤诱导敲低GGPS1后，WB检测P62和LC3B的表达水平。B. After induction of GGPS1 knockdown in H82R tumors, the expression levels of P62 and LC3B were detected by WB.
C.免疫组化检测NCAM、GGPS1、CC3和H2AX染色在H82R肿瘤和诱导敲低GGPS1肿瘤中。统计GGPS1、CC3和H2AX染色在H82R肿瘤和诱导敲低GGPS1肿瘤中的染色比例。比例尺：50μm。C. Immunohistochemical detection of NCAM, GGPS1, CC3 and H2AX staining in H82R tumors and induced knockdown of GGPS1 tumors. The proportion of GGPS1, CC3 and H2AX staining in H82R tumors and induced knockdown of GGPS1 tumors was calculated. Scale bar: 50 μm.
D.H82R诱导敲低RAB7A细胞皮下成瘤后，分别给予对照处理(水，每组五只)和doxycycline(1毫克/毫升溶于饮用水中，每组五只)。肿瘤体积通过游标卡尺测量后计算。图中显示了每组肿瘤增长曲线图(左)，每组肿瘤重量图(中)，肿瘤拍照图(右)。After D.H82R induced knockdown of RAB7A cells subcutaneously, they were given control treatment (water, five animals in each group) and doxycycline (1 mg/ml dissolved in drinking water, five animals in each group). Tumor volume was calculated by vernier caliper measurement. The figure shows the graph of tumor growth in each group (left), the graph of tumor weight in each group (middle), and the graph of tumor photography (right).
E.H82R肿瘤诱导敲低RAB7A后，WB检测P62和LC3B的表达水平。After induction of RAB7A knockdown in E.H82R tumors, the expression levels of P62 and LC3B were detected by WB.
F.免疫组化检测NCAM、RAB7A、CC3和H2AX染色在H82R肿瘤和诱导敲低RAB7A肿瘤中。比例尺：50μm。统计GGPS1、CC3和H2AX染色在H82R肿瘤和诱导敲低RAB7A肿瘤中的染色比例。F. Immunohistochemical detection of NCAM, RAB7A, CC3 and H2AX staining in H82R tumors and induced knockdown of RAB7A tumors. Scale bar: 50 μm. The proportion of GGPS1, CC3 and H2AX staining in H82R tumor and induced knockdown RAB7A tumor was calculated.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图13、Statin抑制GGPS1升高的小细胞肺癌化疗耐药PDX。Figure 13. Statin inhibits chemotherapy-resistant PDX in small cell lung cancer with elevated GGPS1.
A.每一代小细胞肺癌PDX ZS7体内对于E/P药物的效应。对照组(绿色)和化疗处理组(红色)数据以单线条的显示呈现。对小鼠接种PDX敏感肿瘤和耐药肿瘤在给与E/P药物情况下生存分析。A. The effect of each generation of small cell lung cancer PDX ZS7 on E/P drugs in vivo. Control (green) and chemotherapy-treated (red) data are presented as single-line displays. Survival analysis of mice inoculated with PDX-sensitive tumors and drug-resistant tumors in the presence of E/P drugs.
B.每一代小细胞肺癌PDX ZS4体内对于E/P药物的效应。对照组(绿色)和化疗处理组(红色)数据以单线条的显示呈现。对小鼠接种PDX敏感肿瘤和耐药肿瘤在给与E/P药物情况下生存分析。B. The effect of each generation of small cell lung cancer PDX ZS4 on E/P drugs in vivo. Control (green) and chemotherapy-treated (red) data are presented as single-line displays. Survival analysis of mice inoculated with PDX-sensitive tumors and drug-resistant tumors in the presence of E/P drugs.
C.通过WB检测MVA通路基因在PDX肿瘤ZS7和ZS7R，ZS4和ZS4R的表达水平。C. The expression levels of MVA pathway genes in PDX tumors ZS7 and ZS7R, ZS4 and ZS4R were detected by WB.
D.小鼠接种PDX ZS7R后，分为四组，分别按照对照处理，E/P处理，MevaStatin处理和联合处理。肿瘤体积通过游标卡尺测量后计算。图中显示了每组肿瘤增长曲线图(左)，每组肿瘤重量图(右)。D. After inoculation with PDX ZS7R, mice were divided into four groups, which were treated with control, E/P, MevaStatin and combined treatment. Tumor volume was calculated by vernier caliper measurement. Graphs of tumor growth curves for each group (left) and tumor weights for each group (right) are shown.
E.免疫组化检测CC3和H2AX染色在不同处理组下ZS7R肿瘤。统计CC3和H2AX染色在不同处理组下ZS7R肿瘤的染色比例。E. Immunohistochemical detection of CC3 and H2AX staining of ZS7R tumors in different treatment groups. The proportions of ZS7R tumors stained by CC3 and H2AX in different treatment groups were counted.
所有数据分析采用双因素方差分析或者未配对的t检验。生存分析采用log-rank(Mantel–Cox)检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差 采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. Survival analysis was performed using the log-rank (Mantel–Cox) test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图14、Statin抑制高表达GGPS1的PD小细胞肺癌PDX。Figure 14. Statin inhibits PDX in PD small cell lung cancer with high expression of GGPS1.
A.处于PR和PD阶段的小细胞肺癌PDX中GGPS1表达水平代表性组化图。比例尺：50μm。A. Representative histochemical images of GGPS1 expression levels in small cell lung cancer PDX in PR and PD stages. Scale bar: 50 μm.
B.免疫组化检测不同阶段的PR、SD和PD小细胞肺癌PDX肿瘤的GGPS1表达并给予组化打分。B. Immunohistochemical detection of GGPS1 expression in different stages of PR, SD and PD small cell lung cancer PDX tumors and histochemical scoring.
C.通过WB检测GGPS1在不同阶段的PR、SD和PD小细胞肺癌PDX的表达水平。C. The expression levels of GGPS1 in PR, SD and PD small cell lung cancer at different stages were detected by WB.
D.接种PDX SP9后，分别按照对照处理，E/P处理，MevaStatin处理和联合处理。肿瘤体积通过游标卡尺测量后计算。图中显示了每组肿瘤增长曲线图(左)，每组肿瘤重量图(中)，小鼠重量图(右)。D. After inoculation with PDX SP9, follow the control treatment, E/P treatment, MevaStatin treatment and combined treatment respectively. Tumor volume was calculated by vernier caliper measurement. The figure shows a graph of tumor growth in each group (left), a graph of tumor weight in each group (middle), and a graph of mouse weight (right).
E.对取下的肿瘤拍照图。E. Photographs of the removed tumor.
F.不同处理组下，免疫组化检测SP9肿瘤中CC3和H2AX染色。比例尺：50μm。统计CC3和H2AX染色在不同处理组下SP9肿瘤的染色比例。F. Immunohistochemical detection of CC3 and H2AX staining in SP9 tumors under different treatment groups. Scale bar: 50 μm. The proportion of SP9 tumors stained by CC3 and H2AX in different treatment groups was counted.
所有数据分析采用双因素方差分析或者未配对的t检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。All data analyses were performed using two-way ANOVA or unpaired t-test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
图15、通过TCGA分析发现高表达GGPS1的小细胞肺癌患者预后更差。Figure 15. It was found by TCGA analysis that the prognosis of small cell lung cancer patients with high expression of GGPS1 was worse.
A.通过TCGA分析检测香叶基香叶基二磷酸合酶1(GGPS1)的表达水平和小细胞肺癌患者预后关系。A. The relationship between the expression level of geranylgeranyl diphosphate synthase 1 (GGPS1) and the prognosis of patients with small cell lung cancer was detected by TCGA analysis.
B.通过TCGA分析检测鲨烯环氧酶(SQLE)和法尼二磷酸酯法尼基转移酶(FDFT1)的表达水平和小细胞肺癌患者预后关系。生存分析采用log-rank(Mantel-Cox)检验。*p<0.05，**p<0.01，***p<0.001和****p<0.0001。所有误差采用均数标准误差。B. The relationship between the expression levels of squalene epoxidase (SQLE) and farnesyl diphosphate farnesyltransferase (FDFT1) and the prognosis of patients with small cell lung cancer was detected by TCGA analysis. Survival analysis was performed using the log-rank (Mantel-Cox) test. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. All errors are standard error of the mean.
具体实施方式Detailed ways
本发明首次揭示了一种新型的与小细胞肺癌的诊断和治疗密切相关的信号通路：GGPS1/RAB7A/自噬流信号通路。本发明披露了由该信号通路参与的、调控小细胞肺癌及其耐药性的新机制。The present invention discloses for the first time a novel signaling pathway closely related to the diagnosis and treatment of small cell lung cancer: GGPS1/RAB7A/autophagy flow signaling pathway. The invention discloses a new mechanism involved in the signaling pathway to regulate small cell lung cancer and its drug resistance.
小细胞肺癌耐药模型Small cell lung cancer drug resistance model
与大多数肿瘤不同，小细胞肺癌的首选治疗方案是化疗而不是手术，而化疗耐药的快速出现和后续治疗方案的匮乏是导致小细胞肺癌患者预后差、死亡率高的主要原因。其中，耐药后治疗手段匮乏的原因主要在于小细胞肺癌的耐药样本很难获取，使得对其耐药机制的研究及理解严重滞后。代谢异常在肿瘤恶性进展中发挥着重要作用，然而其在小细胞肺癌化疗耐药中的功能并不清楚。因此，建立能够真实模拟临床化疗 耐药的小细胞肺癌模型是解除目前研究困境、解答上述科学问题的关键。Unlike most tumors, chemotherapy rather than surgery is the preferred treatment for SCLC, and the rapid emergence of chemotherapy resistance and the lack of follow-up treatment options are the main reasons for the poor prognosis and high mortality of SCLC patients. Among them, the main reason for the lack of post-resistance treatment methods is that the drug-resistant samples of small cell lung cancer are difficult to obtain, which makes the research and understanding of the drug resistance mechanism seriously lag behind. Metabolic abnormalities play an important role in tumor malignant progression, however, their function in chemoresistance in small cell lung cancer is unclear. Therefore, establishing a small cell lung cancer model that can truly simulate clinical chemoresistance is the key to relieve the current research dilemma and answer the above scientific questions.
目前本领域没有通过体内肿瘤模型建立配对的化疗耐药细胞系，而本发明人成功建立了小细胞肺癌化疗耐药模型。作为本发明的优选方式，小细胞肺癌细胞系(如实施例中称为H82、H209、H526和H146)在体外和体内都对化疗药物(如E/P)敏感。在体内不断给药处理后，得到化疗耐药肿瘤(如实施例中称为H82R、H209R、H526R和H146R)。进一步通过原代培养，成功建立耐药细胞株。At present, there is no paired chemotherapy-resistant cell line established by in vivo tumor models in the art, but the inventors have successfully established a chemotherapy-resistant small cell lung cancer model. As a preferred mode of the present invention, small cell lung cancer cell lines (referred to as H82, H209, H526 and H146 in the examples) are sensitive to chemotherapeutic drugs (eg E/P) both in vitro and in vivo. After continuous administration in vivo, chemotherapy-resistant tumors (referred to as H82R, H209R, H526R and H146R in the examples) were obtained. Further through primary culture, drug-resistant cell lines were successfully established.
作为本发明的优选方式，所述耐药性小细胞肺癌的细胞株的方法包括：(a)将对化疗药物敏感的小细胞肺癌细胞移植于受体；(b)对(a)的受体给予化疗药物处理，使对化疗药物敏感的小细胞肺癌细胞形成的肿瘤发生耐药；较佳地，所述处理包括：对化疗药物敏感的小细胞肺癌细胞给予受体后，肿瘤体积长至约50-400mm 3(较佳地，100-200mm 3)时，给予依托泊苷和顺铂(E/P)化疗药物处理；若给药后肿瘤明显消退则暂停给药，等肿瘤重新恢复生长再继续给药；重复此过程直至化疗药物无法有效抑制肿瘤生长；(c)从(b)的受体的肿瘤中分离出小细胞肺癌细胞，进行原代培养和/或继代培养，获得耐药性小细胞肺癌的细胞株。 As a preferred mode of the present invention, the method for the drug-resistant small cell lung cancer cell line comprises: (a) transplanting small cell lung cancer cells sensitive to chemotherapeutic drugs into a recipient; (b) the recipient of (a) Administering chemotherapeutic drug treatment to make the tumor formed by the chemotherapeutic drug-sensitive small cell lung cancer cells resistant; preferably, the treatment includes: after the chemotherapeutic drug-sensitive small cell lung cancer cells are administered to the receptor, the tumor volume grows to about 50-400mm 3 (preferably, 100-200mm 3 ), give etoposide and cisplatin (E/P) chemotherapy drugs; if the tumor subsides significantly after administration, the administration should be suspended, and the tumor will resume growth again. Continue the administration; repeat this process until the chemotherapeutic drugs cannot effectively inhibit tumor growth; (c) isolate small cell lung cancer cells from the tumor of the recipient of (b), carry out primary culture and/or subculture, and acquire drug resistance Small cell lung cancer cell lines.
在获得该模型的基础上，利用FDA认证的代谢类药物进行药库筛选，发现他汀类药物可以显著抑制化疗耐药小细胞肺癌的存活。On the basis of obtaining this model, the drug library was screened using FDA-certified metabolic drugs, and it was found that statins can significantly inhibit the survival of chemotherapy-resistant small cell lung cancer.
GGPS1/RAB7A/自噬流信号通路及其调控GGPS1/RAB7A/autophagy flow signaling pathway and its regulation
如本发明所用，所述的“(信号)通路”是指由一系列基因或蛋白或其代谢产物(合成产物或加工产物)之间发生相互制约或相互作用而形成的信号系统，也包括通路蛋白与细胞内其它元件或细胞器的相互作用，有时也包括其上下游基因或蛋白的共同参与，其一般会导致一些细胞事件的发生。所述的GGPS1/RAB7A/自噬流信号通路主要包括以下要件：GGPS1基因(和/或其编码的蛋白)、RAB7A基因(和/或其编码的蛋白)、自噬流信号通路。其中，所述的自噬流信号通路包括自噬体与溶酶体的参与。As used in the present invention, the "(signal) pathway" refers to a signal system formed by mutual restriction or interaction between a series of genes or proteins or their metabolites (synthetic products or processing products), and also includes pathways The interaction of a protein with other elements or organelles in the cell, sometimes including the joint participation of its upstream and downstream genes or proteins, generally leads to the occurrence of some cellular events. The GGPS1/RAB7A/autophagy flux signaling pathway mainly includes the following elements: GGPS1 gene (and/or its encoded protein), RAB7A gene (and/or its encoded protein), and autophagy flux signaling pathway. Wherein, the autophagic flow signaling pathway includes the participation of autophagosome and lysosome.
如本发明所用，所述的“(信号)通路”与“(信号)途径”可互换使用。As used herein, the "(signaling) pathway" and "(signaling) pathway" are used interchangeably.
如本发明所用，术语“GGPS1/RAB7A/自噬流信号通路”与“GGPS1-RAB7A-自噬流信号通路”可互换使用。As used herein, the terms "GGPS1/RAB7A/autophagic flux signaling pathway" and "GGPS1-RAB7A-autophagic flux signaling pathway" are used interchangeably.
GGPS1基因的核苷酸序列例如如GenBank_NC_000001.11(人源)所示；其蛋白氨基酸序列例如如GenBank_AAH67768.1(人源)所示。The nucleotide sequence of the GGPS1 gene is shown in, for example, GenBank_NC_000001.11 (human origin); the protein amino acid sequence thereof is shown in, for example, GenBank_AAH67768.1 (human origin).
RAB7A基因的核苷酸序列例如如GenBank_NC_000003.12(人源)所示；其蛋白氨基酸序列例如如GenBank_AAH13728.2(人源)所示。The nucleotide sequence of the RAB7A gene is shown, for example, in GenBank_NC_000003.12 (human origin); the protein amino acid sequence thereof is shown in, for example, GenBank_AAH13728.2 (human origin).
HMGCR基因的核苷酸序列例如如GenBank_NC_000005.10(人源)所示；其蛋白氨基酸序列例如如GenBank_AAH33692.1(人源)所示。The nucleotide sequence of the HMGCR gene is shown, for example, in GenBank_NC_000005.10 (human origin); the protein amino acid sequence thereof is shown in, for example, GenBank_AAH33692.1 (human origin).
本发明中，除非另外说明，涉及MVA信号通路的其它通路蛋白/基因或其上下游蛋白/基因的信息，也是本领域技术人员已知的。In the present invention, unless otherwise specified, information on other pathway proteins/genes of the MVA signaling pathway or their upstream and downstream proteins/genes is also known to those skilled in the art.
上述蛋白(多肽)也包括其变异形式，包括(但并不限于)：若干个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个，还更佳如1-8个、1-5个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。任何与所述蛋白同源性高(比如与所述多肽序列的同源性为70％或更高；优选地同源性为80％或更高；更优选地同源性为90％或更高，如同源性95％，98％或99％)的、且具有蛋白相同功能的蛋白也包括在本发明内。本发明还包括一种突变形式的蛋白或蛋白截短体，只要该突变蛋白或截短体基本上保留了全长蛋白的功能。The above-mentioned protein (polypeptide) also includes its variant forms, including (but not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1- 10, still more preferably such as 1-8, 1-5) amino acid deletions, insertions and/or substitutions, and C-terminal and/or N-terminal additions or deletions of one or more (usually within 20, Preferably within 10, more preferably within 5) amino acids. Any with high homology to the protein (such as 70% or more homology to the polypeptide sequence; preferably 80% or more homology; more preferably 90% or more homology High, such as 95%, 98% or 99% homology) and having the same function as the protein are also included in the present invention. The present invention also includes a mutant form of the protein or protein truncate, so long as the mutant protein or truncate substantially retains the function of the full-length protein.
上述基因的序列也包括与之相简并的序列。编码蛋白的多核苷酸(基因)可以是天然基因，也可以是它们的简并的序列。The sequences of the above-mentioned genes also include degenerate sequences therewith. Polynucleotides (genes) encoding proteins can be native genes or their degenerate sequences.
经过广泛的药物筛选实验，本发明人发现，他汀(Statin)类药物可显著抑制化疗耐药肿瘤的存活。进一步体外和体内实验验证，发现Statin可以抑制多株小细胞肺癌化疗耐药细胞的生长。本发明人发现，甲羟戊酸途径(Mevalonate pathway)下游代谢物GGPP能够完全逆转Statin对于耐药细胞的抑制效应。After extensive drug screening experiments, the inventors found that statins can significantly inhibit the survival of chemotherapy-resistant tumors. Further in vitro and in vivo experiments confirmed that Statin can inhibit the growth of chemotherapy-resistant cells in multiple small cell lung cancers. The inventors found that the downstream metabolite GGPP of the mevalonate pathway can completely reverse the inhibitory effect of Statin on drug-resistant cells.
MVA途径是以乙酰辅酶A为原料合成异戊二烯焦磷酸和二甲烯丙基焦磷酸的一条代谢途径，存在于所有高等真核生物和很多病毒中。该途径的产物是类固醇、类萜等生物分子的合成前体，也是胆固醇合成途径的必经步骤。其下游参与细胞很多重要功能，比如胆固醇合成、线粒体功能、蛋白的异戊二烯化修饰。其中HMGCR是MVA代谢通路的起始和限速步骤，它会合成甲羟戊酸，该代谢酶可由他汀药物所抑制。之后会到下游产物GPP，经FDPS合成FPP，FPP会经FDFT1合成为鲨烯，鲨烯会由SQLE继续合成胆固醇。除此之外，FPP还可以经GGPS1合成GGPP，GGPP可对靶蛋白进行香叶基香叶基化修饰，这类修饰属于异戊二烯类修饰，在细胞内发挥着重要功能。The MVA pathway is a metabolic pathway for the synthesis of isoprene pyrophosphate and dimethylallyl pyrophosphate from acetyl-CoA, which exists in all higher eukaryotes and many viruses. The products of this pathway are the synthetic precursors of biomolecules such as steroids and terpenoids, and are also necessary steps in the cholesterol synthesis pathway. Its downstream is involved in many important functions of cells, such as cholesterol synthesis, mitochondrial function, and protein prenylation modification. Among them, HMGCR is the initial and rate-limiting step of the MVA metabolic pathway, which synthesizes mevalonate, a metabolic enzyme that can be inhibited by statins. After that, it will go to the downstream product GPP, FPP will be synthesized by FDPS, FPP will be synthesized into squalene through FDFT1, and squalene will continue to synthesize cholesterol by SQLE. In addition, FPP can also synthesize GGPP through GGPS1, and GGPP can carry out geranylgeranylation modification of target protein, which belongs to isoprene modification and plays an important function in cells.
本发明人研究发现，补充甲羟戊酸途径的甲羟戊酸(MVA)或其下游中间代谢物香叶基香叶基焦磷酸(GGPP)可以有效阻断他汀类药物对化疗耐药小细胞肺癌的杀伤。深入分析揭示，他汀类药物(Statin)主要是通过抑制MVA代谢途径中GGPP的产生，干扰了GGPP对小G蛋白RAB7A进行香叶基香叶基化修饰，使后者无法上膜发挥正常功能，进而抑制了自噬体与溶酶体的融合，引发自噬流障碍，最终导致化疗耐药细胞死亡。也即，Statin由GGPS1-RAB7A-Autophagic途径(自噬流信号通路)介导、可导致自噬流障碍，引发细胞死亡，发挥抑制耐药癌细胞存活效应的。The inventors have found that mevalonate (MVA) supplementing the mevalonate pathway or its downstream intermediate metabolite geranylgeranyl pyrophosphate (GGPP) can effectively block the effect of statins on chemotherapy-resistant small cells The killing of lung cancer. In-depth analysis revealed that statins mainly inhibit the production of GGPP in the MVA metabolic pathway, interfering with the geranylgeranylation modification of the small G protein RAB7A by GGPP, so that the latter cannot perform normal functions on the membrane. This in turn inhibits the fusion of autophagosomes and lysosomes, triggering autophagic flux barriers, and ultimately leading to the death of chemotherapy-resistant cells. That is, Statin is mediated by the GGPS1-RAB7A-Autophagic pathway (autophagic flow signaling pathway), which can lead to autophagic flow barriers, trigger cell death, and inhibit the survival of drug-resistant cancer cells.
如本发明所用，所述的“抑制(剂)”与“下调(剂)”可互换使用，其也包括：阻滞(剂)，拮抗(剂)等。As used in the present invention, the "inhibitor (agent)" and "down-regulating (agent)" can be used interchangeably, which also include: block (agent), antagonist (agent) and the like.
本发明人发现，所述的信号通路中，下调GGPS1的表达或活性、下调RAB7A的 膜定位或下调GGPS1代谢产物GGPP对RAB7A的修饰作用，可以抑制小细胞肺癌。因此，可以通过这种作用模式，来筛选或设计适用于靶向调控的药物。The inventors found that in the signaling pathway, down-regulating the expression or activity of GGPS1, down-regulating the membrane localization of RAB7A or down-regulating the modification effect of GGPS1 metabolite GGPP on RAB7A can inhibit small cell lung cancer. Therefore, this mode of action can be used to screen or design drugs suitable for targeted regulation.
应理解，在得知了所述GGPS1/RAB7A/自噬流信号通路(较佳地，也包括其上下游蛋白或基因)的功能后，可以采用本领域人员熟知的多种方法来调节所述的GGPS1/RAB7A/自噬流信号通路。比如可以采用本领域人员熟知的多种方法来调节通路蛋白的表达或使之缺失表达。或采用本领域技术人员熟知的方法来促进或减弱自噬流的发生。It should be understood that after knowing the function of the GGPS1/RAB7A/autophagy flow signaling pathway (preferably, also including its upstream and downstream proteins or genes), various methods well known to those in the art can be used to regulate the The GGPS1/RAB7A/autophagic flow signaling pathway. For example, various methods well known to those skilled in the art can be used to modulate or deplete the expression of pathway proteins. Or use methods well known to those skilled in the art to promote or attenuate the occurrence of autophagic flux.
作为本发明的优选方式，提供了下调GGPS1的表达或活性、下调RAB7A的膜定位或下调GGPS1代谢产物GGPP对RAB7A的修饰作用的下调剂。所述下调剂是指任何可降低GGPS1或RAB7A的活性、降低GGPS1或RAB7A的稳定性、下调GGPS1或RAB7A的表达、减少GGPS1或RAB7A有效作用时间、抑制GGPS1或RAB7A的转录和翻译的物质的物质，这些物质均可用于本发明，作为对于抑制小细胞肺癌潜在有用的物质。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。例如，所述的下调剂是：特异性干扰GGPS1或RAB7A或其上游基因表达的干扰RNA分子或反义核苷酸；或是特异性编辑GGPS1或RAB7A或其上游基因编辑试剂，等等。As a preferred mode of the present invention, there is provided a down-regulating agent that down-regulates the expression or activity of GGPS1, down-regulates the membrane localization of RAB7A, or down-regulates the modification effect of GGPS1 metabolite GGPP on RAB7A. The down-regulating agent refers to any substance that can reduce the activity of GGPS1 or RAB7A, reduce the stability of GGPS1 or RAB7A, down-regulate the expression of GGPS1 or RAB7A, reduce the effective time of GGPS1 or RAB7A, and inhibit the transcription and translation of GGPS1 or RAB7A. , these substances can be used in the present invention as potentially useful substances for inhibiting small cell lung cancer. They can be chemical compounds, small chemical molecules, biomolecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level. For example, the down-regulating agents are: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of GGPS1 or RAB7A or their upstream genes; or specifically editing GGPS1 or RAB7A or their upstream gene editing agents, and so on.
本发明提供了一种下调GGPS1/RAB7A/自噬流信号通路的方法，包括对GGPS1/RAB7A/自噬流信号通路中GGPS1或RAB7A基因进行靶向性地突变、基因编辑或基因重组，从而实现下调。作为一种更为具体的实施例方式，藉由上述任一的方法，可使GGPS1或RAB7A转变为其功能丧失的截短体或突变体。作为一种更为具体的可实施方式，采用CRISPR/Cas系统进行基因编辑，从而敲除或下调靶基因。合适的sgRNA靶位点，会带来更高的基因编辑效率，所以在着手进行基因编辑前，可以设计并找到合适的靶位点。在设计特异性靶位点后，还需要进行体外细胞活性筛选，以获得有效的靶位点用于后续实验。The present invention provides a method for down-regulating the GGPS1/RAB7A/autophagy flow signal pathway, including targeted mutation, gene editing or gene recombination of the GGPS1 or RAB7A gene in the GGPS1/RAB7A/autophagy flow signal pathway, so as to achieve down. As a more specific embodiment, GGPS1 or RAB7A can be converted into a loss-of-function truncation or mutant thereof by any of the above methods. As a more specific embodiment, the CRISPR/Cas system is used for gene editing, thereby knocking out or down-regulating the target gene. Appropriate sgRNA target sites will bring higher gene editing efficiency, so before proceeding with gene editing, suitable target sites can be designed and found. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments.
作为本发明的另一种实施方式，提供了一种下调GGPS1或RAB7A的表达的方法，包括：将干扰GGPS1或RAB7A基因表达的干扰分子转入细胞，或通过合适的途径处理细胞、使之被引入到细胞内，例如设计穿膜功能域使之具有穿膜的能力。As another embodiment of the present invention, there is provided a method for down-regulating the expression of GGPS1 or RAB7A, comprising: transferring an interfering molecule that interferes with the expression of GGPS1 or RAB7A into cells, or treating cells by appropriate means to make them Introduced into cells, such as designing the transmembrane domain to have the ability to transmembrane.
当用于作为人工调控的靶标时或人为建立筛选系统时，以上的蛋白或编码基因可以是天然存在的，比如其可被纯化和分离自哺乳动物；也可以是重组制备的，比如可以根据常规的基因重组技术来生产重组的蛋白。此外，任何不影响这些蛋白的生物活性的变化形式都是可用的，如它们的功能未发生改变的衍生物或变异体。When used as a target for artificial regulation or an artificially established screening system, the above proteins or coding genes may be naturally occurring, for example, they may be purified and isolated from mammals; they may also be recombinantly prepared, for example, according to conventional genetic recombination technology to produce recombinant proteins. In addition, any variation that does not affect the biological activity of these proteins is available, such as derivatives or variants that do not alter their function.
基于GGPS1/RAB7A/自噬流信号通路的药物筛选Drug screening based on GGPS1/RAB7A/autophagy flux signaling pathway
基于本发明人的新发现，对GGPS1/RAB7A/自噬流信号通路的研究有着多方面的用途，所述的用途包括：筛选调节该信号通路的物质，以期用于抑制小细胞肺癌。其中，所述的调节包括：下调GGPS1的表达或活性，下调RAB7A的膜定位，下调GGPS1代谢产物GGPP对RAB7A的修饰作用，或促进自噬流障碍等。Based on the new findings of the present inventors, the research on the GGPS1/RAB7A/autophagy flow signaling pathway has various uses, and the uses include: screening for substances that regulate this signaling pathway, in order to inhibit small cell lung cancer. Wherein, the regulation includes: down-regulating the expression or activity of GGPS1, down-regulating the membrane localization of RAB7A, down-regulating the modification effect of GGPS1 metabolite GGPP on RAB7A, or promoting autophagic flow disorder, etc.
本发明提供了一种筛选调节GGPS1/RAB7A/自噬流信号通路的调节剂方法，通过向含有GGPS1/RAB7A/自噬流信号通路的体系中添加待筛选的候选物，并观察GGPS1/RAB7A/自噬流信号通路中各蛋白或基因的变化或相互作用来进行筛选。若候选物质下调GGPS1的表达或活性，下调RAB7A的膜定位，下调GGPS1代谢产物GGPP对RAB7A的修饰作用，或促进自噬流障碍，则该候选物质是对于抑制小细胞肺癌有用的物质。The present invention provides a method for screening regulators regulating GGPS1/RAB7A/autophagy flow signaling pathway, by adding candidates to be screened to a system containing GGPS1/RAB7A/autophagy flow signaling pathway, and observing GGPS1/RAB7A/ The changes or interactions of each protein or gene in the autophagic flow signaling pathway were screened. If the candidate substance down-regulates the expression or activity of GGPS1, down-regulates the membrane localization of RAB7A, down-regulates the modification effect of GGPS1 metabolite GGPP on RAB7A, or promotes the disturbance of autophagic flow, the candidate substance is a useful substance for inhibiting small cell lung cancer.
如本文所用，所述的“抑制”、“提高”、“促进”均是指具有统计学意义的“抑制”、“提高”、“促进”。也即：显著地“抑制”、“提高”、“促进”。如与对照组的蛋白活性、蛋白表达、蛋白结合或甲基化程度相比，显著“抑制”、“提高”、“促进”10％，20％，30％，40％，50％以上；更佳的60％，70％，80％以上。As used herein, the terms "inhibit", "increase" and "promote" all refer to "inhibit", "increase" and "promote" with statistical significance. That is: significantly "inhibit", "increase", "promote". For example, compared with the protein activity, protein expression, protein binding or methylation degree of the control group, significantly "inhibit", "increase", "promote" 10%, 20%, 30%, 40%, 50% or more; more Best 60%, 70%, 80% or more.
所述的含有GGPS1/RAB7A/自噬流信号通路的体系选自：细胞体系(或细胞培养物体系)、亚细胞体系(或亚细胞培养物体系)、溶液体系、动物体系或组织体系(或组织培养物体系)。The system containing GGPS1/RAB7A/autophagy flow signaling pathway is selected from: cell system (or cell culture system), subcellular system (or subcellular culture system), solution system, animal system or tissue system (or tissue culture system).
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以从候选物质中进一步选择和确定对于抑制小细胞肺癌有用的物质。As a preferred mode of the present invention, the method further includes: performing further cell experiments and/or animal experiments on the obtained potential substances to further select and determine the substances useful for inhibiting small cell lung cancer from the candidate substances.
当进行筛选时，可以采用本领域熟知的各种技术来确定蛋白或其编码基因的变化情况以及相互作用情况。When screening, various techniques well known in the art can be used to determine changes and interactions of proteins or their encoding genes.
可以采用多种常规的技术来鉴定系统中基因的转录或表达情况。这些技术包括但不限于：寡核苷酸杂交技术(如探针)，多聚酶链反应(PCR)，聚丙烯酰胺凝胶电泳等。检测蛋白与蛋白之间相互作用以及相互作用的强弱可采用多种本领域技术人员熟知的技术，比如免疫共沉淀技术、GST沉降技术、噬菌体展示技术或酵母双杂交系统。蛋白的核定位也是本领域熟知的技术。A variety of conventional techniques can be used to identify transcription or expression of genes in the system. These techniques include, but are not limited to, oligonucleotide hybridization techniques (eg, probes), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and the like. To detect the interaction between proteins and the strength of the interaction, various techniques well known to those skilled in the art can be used, such as co-immunoprecipitation technique, GST sedimentation technique, phage display technique or yeast two-hybrid system. Nuclear localization of proteins is also a technique well known in the art.
通过上述方法初步筛选出的物质可构成一个筛选库，以便于人们最终可以从中筛选出能够对于抑制小细胞肺癌真正有用的物质。Substances preliminarily screened by the above method can constitute a screening library, so that people can finally screen out substances that are really useful for inhibiting small cell lung cancer.
本发明还提供了采用所述筛选方法获得的可用于抑制小细胞肺癌的潜在物质。The present invention also provides potential substances obtained by the screening method which can be used to inhibit small cell lung cancer.
本发明还提供了一种制备抑制小细胞肺癌(特别是抑制小细胞肺癌)的药物的方法，所述方法包括：合成和/或纯化前述筛选获得的对于抑制小细胞肺癌有用的物质，作为用于抑制小细胞肺癌的药物。The present invention also provides a method for preparing a drug for inhibiting small cell lung cancer (especially for inhibiting small cell lung cancer), the method comprising: synthesizing and/or purifying the substances obtained by the aforementioned screening that are useful for inhibiting small cell lung cancer, as Drugs for the inhibition of small cell lung cancer.
可将获得的对于抑制小细胞肺癌有用的物质用于制备药物组合物，如本发明的下文中所述。The obtained substances useful for inhibiting small cell lung cancer can be used for the preparation of pharmaceutical compositions, as hereinafter described in the present invention.
以蛋白或基因或其上特定的区域作为靶点，来筛选作用于该靶点的物质的方法是本领域人员所熟知的，这些方法均可用于本发明。所述的候选物质可以选自：肽、聚合肽、拟肽、非肽化合物、碳水化合物、脂、抗体或抗体片段、配体、有机小分子、无机小分子和核酸序列等。根据待筛选的物质的种类，本领域人员清楚如何选择适用的筛选方法。The methods for screening substances acting on the target by taking a protein or gene or a specific region on it as a target are well known to those skilled in the art, and these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the type of substances to be screened, it is clear to those skilled in the art how to select a suitable screening method.
联合用药Combination medication
本发明人发现，他汀类药物、依托泊苷和顺铂的联合用药，能够极其显著地增强小细胞肺癌的抑制效果。他汀类药物与依托泊苷和顺铂的协同作用藉由以下作用方式：他汀类药物抑制MVA代谢途径中GGPP的产生，干扰GGPP对小G蛋白RAB7A进行香叶基香叶基化修饰，使后者无法上膜发挥正常功能，进而抑制了自噬体与溶酶体的融合，引发自噬流障碍，最终导致化疗耐药细胞死亡。本发明中，下调GGPS1，可以进一步地减少GGPP对小G蛋白RAB7A进行香叶基香叶基化修饰，也可直接下调RAB7A，进而抑制自噬体与溶酶体的融合，引发自噬流障碍。The inventors found that the combination of statins, etoposide and cisplatin can significantly enhance the inhibitory effect of small cell lung cancer. The synergistic effect of statins with etoposide and cisplatin is through the following modes of action: statins inhibit the production of GGPP in the MVA metabolic pathway, interfere with the geranylgeranylation modification of the small G protein RAB7A by GGPP, and make the later The cells are unable to function normally on the upper membrane, which in turn inhibits the fusion of autophagosomes and lysosomes, triggers autophagic flow barriers, and eventually leads to the death of chemotherapy-resistant cells. In the present invention, down-regulating GGPS1 can further reduce the geranylgeranylation modification of small G protein RAB7A by GGPP, or directly down-regulate RAB7A, thereby inhibiting the fusion of autophagosome and lysosome, and causing autophagic flow disorder .
基于本发明人的新发现，本发明提供了一种抑制小细胞肺癌的药物组合物，包括药物组合：他汀类药物、依托泊苷和顺铂。Based on the new findings of the present inventors, the present invention provides a pharmaceutical composition for inhibiting small cell lung cancer, including a drug combination: statins, etoposide and cisplatin.
如本发明所述，所述的“抑制小细胞肺癌”包括了“降低(逆转)小细胞肺癌的耐药性”。优选地，所述的小细胞肺癌为耐药性的小细胞肺癌。According to the present invention, "inhibiting small cell lung cancer" includes "reducing (reversing) the drug resistance of small cell lung cancer". Preferably, the small cell lung cancer is drug-resistant small cell lung cancer.
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的如本文所用。As used herein, the term "effective amount" or "effective dose" refers to that which is functional or active in humans and/or animals and which is acceptable to humans and/or animals as used herein.
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, a substance with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
本发明也提供了一种用于抑制肿瘤或降低肿瘤耐药性的药盒，所述的药盒中包括：他汀类药物、依托泊苷和顺铂。更优选地，所述药盒中还包括：使用说明书，以指导临床医师以正确合理的方式用药。The present invention also provides a kit for inhibiting tumor or reducing drug resistance of tumor, the kit includes statins, etoposide and cisplatin. More preferably, the kit also includes: instructions for use to guide the clinician to use the medicine in a correct and reasonable manner.
为了方便给药，所述的他汀类药物、依托泊苷和顺铂被混合，制成单元剂型的形式，置于试剂盒中。“单元剂型”是指为了服用方便，将药物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。此外，所述的他汀类药物、依托泊苷和顺铂也可被独立地分置于不同的容器中，在需要时进行混合，应用。For ease of administration, the statin, etoposide, and cisplatin are combined and placed in a kit in unit dosage form. "Unit dosage form" refers to the preparation of a drug into a single dosage form for the convenience of taking, including but not limited to various solid dosage forms (eg, tablets), liquid dosage forms, capsules, and sustained-release dosage forms. In addition, the statins, etoposide and cisplatin can also be separately placed in different containers, mixed when necessary, and applied.
作为本发明的优选方式，所述的他汀类药物、依托泊苷和顺铂的用量比例为(5～ 15)：(1～3)：1；较佳地为(7～10)：(1.5～2.5)：1(如8.6：1.7：1)。As a preferred mode of the present invention, the dosage ratio of the statins, etoposide and cisplatin is (5-15):(1-3):1; preferably (7-10):(1.5) ~2.5): 1 (eg 8.6: 1.7: 1).
作为本发明的优选方式，所述顺铂、依托泊苷和所述他汀类药物的用药方法为：按1周1个疗程计算，根据个体体重，通过腹腔注射方式第1天给予0.5～10毫克/千克(如0.8～6毫克/千克，更具体如1、2、3、4、5毫克/千克)顺铂(CDDP)；第1～3天给予0.5～15毫克/千克/天(如1～12毫克/千克/天，更具体如2、4、6、8、10毫克/千克/天)依托泊苷(VP16)；同时通过灌胃方式给予2～100毫克/千克/天(如5～60毫克/千克/天，更具体如6、8、10、15、20、30、40、50毫克/千克/天)的美伐他汀或辛伐他汀或匹伐他汀等药物处理。As a preferred mode of the present invention, the method of administration of the cisplatin, etoposide and the statin is as follows: one course of treatment per week, according to individual body weight, 0.5-10 mg is given by intraperitoneal injection on the first day /kg (eg 0.8-6 mg/kg, more specifically such as 1, 2, 3, 4, 5 mg/kg) cisplatin (CDDP); 0.5-15 mg/kg/day (eg 1 to 3 days) ~12 mg/kg/day, more specifically 2, 4, 6, 8, 10 mg/kg/day) etoposide (VP16); concurrently 2 to 100 mg/kg/day (eg 5 mg/kg/day) by gavage ~60 mg/kg/day, more specifically such as 6, 8, 10, 15, 20, 30, 40, 50 mg/kg/day) of mevastatin or simvastatin or pitavastatin and other drug treatments.
在本发明的具体实施例中，所述的个体为小鼠，按1周1个疗程计算，根据小鼠体重按照10毫克/千克/天第1，2，3天给予依托泊苷(VP16)，按6毫克/千克/天在第1天给予顺铂(CDDP)，通过腹腔注射的方式给予药物处理。他汀类药物根据小鼠体重按照50毫克/千克/天通过灌胃给予美伐他汀，辛伐他汀，匹伐他汀等药物处理。In a specific embodiment of the present invention, the individual is a mouse, and according to the calculation of one course of treatment per week, etoposide (VP16) is administered at 10 mg/kg/day on the 1st, 2nd, and 3rd days according to the body weight of the mouse. , Cisplatin (CDDP) was administered at 6 mg/kg/day on day 1, and drug treatment was administered by intraperitoneal injection. Statins were administered by gavage at 50 mg/kg/day according to the body weight of mice, such as mevastatin, simvastatin, pitavastatin and other drugs.
尽管本发明的具体实施例中，给出了针对动物如小鼠的给药方案。但应理解，从动物如鼠的给药剂量换算为适用于人类的给药剂量是本领域技术人员易于作出的，例如可根据Meeh-Rubner公式来进行计算：Meeh-Rubner公式：A＝k×(W 2/3)/10,000。式中A为体表面积，以m 2计算；W为体重，以g计算；K为常数，随动物种类而不同，一般而言，小鼠和大鼠9.1，豚鼠9.8，兔10.1，猫9.9，狗11.2，猴11.8，人10.6。应理解，根据药物以及临床情形的不同，根据有经验的药师的评估，给药剂量的换算是可以变化的。 Although specific examples of the present invention are given for dosing regimens for animals such as mice. However, it should be understood that it is easy for those skilled in the art to convert the dose for animals such as mice into the dose suitable for humans. For example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A=k× (W 2/3 )/10,000. In the formula, A is the body surface area, calculated in m2 ; W is the body weight, calculated in g; K is a constant, which varies with animal species. Dog 11.2, Monkey 11.8, Human 10.6. It will be appreciated that, depending on the drug and the clinical situation, the conversion of the administered dose may vary according to the assessment of an experienced pharmacist.
肿瘤诊断或预后评估的应用Application of tumor diagnosis or prognostic assessment
本发明摁发现，高表达GGPS1的化疗耐药PDX小鼠模型对他汀类药物非常敏感，而他汀与化疗联用可以达到更好的效果。临床数据分析揭示，高表达GGPS1的小细胞肺癌患者预后往往更差。因此，GGPS1可作为指导耐药患者给予Statin治疗的潜在分子标志物。The present invention has found that the chemotherapy-resistant PDX mouse model with high expression of GGPS1 is very sensitive to statins, and the combination of statins and chemotherapy can achieve better effects. Analysis of clinical data revealed that SCLC patients with high expression of GGPS1 tended to have worse prognosis. Therefore, GGPS1 may serve as a potential molecular marker to guide the administration of Statin in drug-resistant patients.
本发明人也利用TCGA的数据分析临床样本中GGPS1表达和患者预后的关系，发现高表达GGPS1患者的预后更差。The inventors also used TCGA data to analyze the relationship between GGPS1 expression in clinical samples and patient prognosis, and found that patients with high expression of GGPS1 had a worse prognosis.
基于本发明人的上述新发现，可以将GGPS1作为小细胞肺癌的诊断或预后的标志物，尤其适用于作为化疗后阶段的预后评估的标志物：(i)进行癌症化疗后阶段的疾病分型、鉴别诊断、和/或无病生存率分析；(ii)评估相关人群的肿瘤治疗药物、药物疗效、预后，以及选择合适的治疗方法。比如，可分离出GGPS1基因表达异常的人群，从而可进行更有针对性地治疗。Based on the above-mentioned new findings of the present inventors, GGPS1 can be used as a marker for the diagnosis or prognosis of small cell lung cancer, especially as a marker for prognostic assessment in the post-chemotherapy stage: (i) Disease classification in the post-chemotherapy stage of cancer , differential diagnosis, and/or analysis of disease-free survival; (ii) evaluation of tumor treatment drugs, drug efficacy, prognosis, and selection of appropriate treatment methods in relevant populations. For example, people with abnormal GGPS1 gene expression can be isolated, allowing for more targeted treatment.
可以通过判断待评估样本中GGPS1的表达情况或活性情况，来预测提供该待评估样本的受试者的疾病预后情况，选择合适的药物实施治疗。通常，可以规定一个GGPS1的 阈值，当GGPS1的表达情况高于所规定的阈值时，考虑采用抑制GGPS1的方案进行治疗。所述的阈值对于本领域技术人员而言是易于确定的，例如可以通过将小细胞肺癌患者的一般GGPS1的表达情况或正常健康人的表达情况进行比较和分析后，获得GGPS1表达异常的阈值。By judging the expression or activity of GGPS1 in the sample to be evaluated, the disease prognosis of the subject who provides the sample to be evaluated can be predicted, and an appropriate drug can be selected for treatment. Usually, a threshold value of GGPS1 can be specified, and when the expression of GGPS1 is higher than the specified threshold value, a regimen of suppressing GGPS1 should be considered for treatment. The threshold can be easily determined by those skilled in the art. For example, the threshold for abnormal GGPS1 expression can be obtained by comparing and analyzing the general GGPS1 expression in patients with small cell lung cancer or the expression in normal healthy people.
因此，本发明提供了GGPS1基因或蛋白的用途，用于制备小细胞肺癌预后评估的试剂或试剂盒。可采用各种本领域已知的技术来检测GGPS1基因或蛋白的存在与否以及表达情况，这些技术均包含在本发明中。例如可用已有的技术如Southern印迹法、Western印迹法、DNA序列分析、PCR等，这些方法可结合使用。本发明还提供了用于在分析物中检测GGPS1基因或蛋白的存在与否以及表达情况的试剂。优选的，当进行基因水平的检测时，可以采用特异性扩增GGPS1的引物；或特异性识别GGPS1的探针来确定GGPS1基因的存在与否；当进行蛋白水平的检测时，可以采用特异性结合GGPS1蛋白的抗体或配体来确定GGPS1蛋白的表达情况。Therefore, the present invention provides the use of the GGPS1 gene or protein for preparing a reagent or kit for evaluating the prognosis of small cell lung cancer. The presence or absence and expression of GGPS1 gene or protein can be detected by various techniques known in the art, and these techniques are all included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination. The present invention also provides reagents for detecting the presence or absence and expression of the GGPS1 gene or protein in an analyte. Preferably, when detecting at the gene level, primers that specifically amplify GGPS1 can be used; or a probe that specifically recognizes GGPS1 can be used to determine the presence or absence of the GGPS1 gene; when detecting the protein level, specific primers can be used. The expression of GGPS1 protein is determined by binding the antibody or ligand of GGPS1 protein.
所述的试剂盒中还可包括用于提取DNA、PCR、杂交、显色等所需的各种试剂，包括但不限于：抽提液、扩增液、杂交液、酶、对照液、显色液、洗液等。此外，所述的试剂盒中还可包括使用说明书和/或核酸序列分析软件等。The kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, display solution, etc. Color solution, lotion, etc. In addition, the kit may also include instructions for use and/or nucleic acid sequence analysis software, and the like.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.
1.材料和试剂1. Materials and Reagents
DH5α感受态(E.coli)；质粒小提试剂盒(Generay)；质粒中提试剂盒(TIANGEN)；DMEM基础培养基(货号：SH30243.01B)和RPMI 1640基础培养基(货号：SH30809.01B)，均购自HyClone公司；胎牛血清(FBS)：货号：S0615，500ml/瓶，购自德国Biochrom AG公司，40ml/管分装后-20℃保存；青霉素、链霉素储存液：货号：15140122，100ml/瓶，购自Invitrogen公司；RevertAidTM First Strand cDNA Synthesis Kit(购自美国Fermentas公司，货号K1622)，SYBR Green Realtime PCR Master Mix(购自美国TOYOBO公司，货号TY-QPK-201)。BABL/C裸鼠，购自上海必凯公司(BK)。SCID小鼠，购自中科院斯莱克实验动物中心(SLAC)。DH5α Competent (E.coli); Plasmid Mini Kit (Generay); Plasmid Intermediate Kit (TIANGEN); DMEM basal medium (Cat. No. SH30243.01B) and RPMI 1640 basal medium (Cat. No. SH30809.01B ), both purchased from HyClone Company; Fetal Bovine Serum (FBS): Item No.: S0615, 500ml/bottle, purchased from Biochrom AG, Germany, 40ml/tube and stored at -20°C; Penicillin, Streptomycin storage solution: Item No. : 15140122, 100ml/bottle, purchased from Invitrogen company; RevertAidTM First Strand cDNA Synthesis Kit (purchased from U.S. Fermentas company, article number K1622), SYBR Green Realtime PCR Master Mix (purchased from U.S. TOYOBO company, article number TY-QPK-201). BABL/C nude mice were purchased from Shanghai Bikai Company (BK). SCID mice were purchased from Slack Laboratory Animal Center (SLAC), Chinese Academy of Sciences.
2.质粒2. Plasmids
pLKO.1-U6-puro购自Addgene。先将载体用AgeI/EcoRI双酶切，将针对目的基因中5′-UTR，CDS或3′-UTR序列中的21个碱基序列加上XhoI酶切位点再加上其反向 互补序列插入到该载体中。慢病毒包装质粒：PSPA和PMD2.G。逆转录病毒包装质粒：pCL10A。pBabe-mCherry-GFP-Puro质粒由美国纪念斯隆凯特琳癌症中心提供。pLKO.1-U6-Tet-on-puro购自Addgene，可以条件性地敲低。使用试剂盒完成质粒的小提中量(TIANGEN，DP107)和质粒的大量制备(TIANGEN，DP117)。pLKO.1-U6-puro was purchased from Addgene. Firstly, the vector is digested with AgeI/EcoRI double enzyme, and the 21-nucleotide sequence in the 5'-UTR, CDS or 3'-UTR sequence of the target gene is added to the XhoI restriction site and its reverse complement is added. inserted into the vector. Lentiviral packaging plasmids: PSPA and PMD2.G. Retroviral packaging plasmid: pCL10A. The pBabe-mCherry-GFP-Puro plasmid was provided by Memorial Sloan Kettering Cancer Center. pLKO.1-U6-Tet-on-puro was purchased from Addgene and can be knocked down conditionally. Minimal extraction of plasmids (TIANGEN, DP107) and large-scale preparation of plasmids (TIANGEN, DP117) were accomplished using kits.
用于敲低GGPS1的shR NA序列为 CCTGAGCTAGTAGCCTTAGTA： The shRNA sequence used to knock down GGPS1 was C CTGAGCTAGTAGCCTTAGTA :
引物序列为shhGGPS1-CDS1-F：The primer sequence is shhGGPS1-CDS1-F:
5’-ccggcctgagctagtagccttagtactcgagtactaaggctactagctcaggtttttg-3’(SEQ ID NO:1)；5'-ccggcctgagctagtagccttagtactcgagtactaaggctactagctcaggtttttg-3' (SEQ ID NO: 1);
shhGGPS1-CDS1-R：shhGGPS1-CDS1-R:
5’-aattcaaaaacctgagctagtagccttagtactcgagtactaaggctactagctcagg-3’(SEQ ID NO:2)。5'-aattcaaaaacctgagctagtagccttagtactcgagtactaaggctactagctcagg-3' (SEQ ID NO:2).
用于敲低RAB7A的shRNA序列为：ggctagtcacaatgcagatat(SEQ ID NO:3)；The shRNA sequence for knocking down RAB7A is: ggctagtcacaatgcagatat (SEQ ID NO:3);
引物序列为shhRAB7A-CDS1-F：The primer sequence is shhRAB7A-CDS1-F:
5’-ccggggctagtcacaatgcagatatctcgagatatctgcattgtgactagcctttttg-3’(SEQ ID NO:4)；5'-ccggggctagtcacaatgcagatatctcgagatatctgcattgtgactagcctttttg-3' (SEQ ID NO:4);
shhRAB7A-CDS1-R：shhRAB7A-CDS1-R:
5’-aattcaaaaaggctagtcacaatgcagatatctcgagatatctgcattgtgactagcc-3’(SEQ ID NO:5)。5'-aattcaaaaaggctagtcacaatgcagatatctcgagatatctgcattgtgactagcc-3' (SEQ ID NO:5).
3.细胞3. Cells
小细胞肺癌细胞株H146，H196，DMS114细胞，购自ATCC，培养条件RPMI medium(Hyclone)+8％FBS+1×P/S。小细胞肺癌细胞株H82，H209细胞，由天津医科大学提供，培养条件RPMI medium(Hyclone)+8％FBS+1×P/S。小细胞肺癌细胞株H446，H526细胞，由上海交通大学提供，培养条件RPMI(Hyclone)配基+8％FBS+1×P/S。Small cell lung cancer cell lines H146, H196, DMS114 cells, purchased from ATCC, culture conditions RPMI medium (Hyclone)+8%FBS+1×P/S. Small cell lung cancer cell lines H82, H209 cells, provided by Tianjin Medical University, culture conditions RPMI medium (Hyclone) + 8% FBS + 1 × P/S. Small cell lung cancer cell lines H446 and H526 cells were provided by Shanghai Jiaotong University, and the culture conditions were RPMI (Hyclone) ligand + 8% FBS + 1×P/S.
悬浮细胞取出细胞于倒置显微镜下观察密度，吸取培养基，800rpm 3min离心后，去上清，1×PBS洗一篇，800rpm 3min离心后，去上清后加入新鲜配基重悬，用1mL移液器将细胞吹散至单细胞后，按照一定比例(一般1：3或1：4)传入新的培养皿中。用十字形摇匀细胞后置于37℃培养箱。Remove the cells from the suspended cells, observe the density under an inverted microscope, aspirate the medium, centrifuge at 800 rpm for 3 min, remove the supernatant, wash one with 1×PBS, centrifuge at 800 rpm for 3 min, remove the supernatant, add fresh ligand, and resuspend with 1 mL. After the liquid container blows the cells to a single cell, it is transferred into a new culture dish according to a certain ratio (generally 1:3 or 1:4). Shake cells in a cross shape and place in a 37°C incubator.
贴壁细胞取出细胞于倒置显微镜下观察密度，一般地，密度大于70％适于传代。传代时，先吸去培养液，加入2mL 1×PBS(10cm Dish)洗两遍，再以1ml/25cm 2面积的量加入胰酶消化液，上下左右摇匀使其均匀覆盖细胞，放入37℃培箱消化，间隙取出在显微镜下观察细胞变圆即可加入适量的培养基终止消化，用1mL移液器将细胞吹散至单细胞后，按照一定比例(一般1：3或1：4)传入新的培养皿中。用十字形摇匀细胞后置于37℃培养箱。 Adherent cells were taken out and the density was observed under an inverted microscope. Generally, the density greater than 70% was suitable for passage. When subculture, first aspirate the culture medium, add 2mL of 1×PBS (10cm Dish) to wash twice, then add trypsin digestion solution in an amount of 1ml/ 25cm2 area, shake up and down, left and right to evenly cover the cells, put in 37 ℃ incubator digestion, take out the gap and observe the cells become rounded under the microscope, then add an appropriate amount of medium to terminate the digestion, blow the cells to single cells with a 1mL pipette, and follow a certain ratio (usually 1:3 or 1:4). ) into a new petri dish. Shake cells in a cross shape and place in a 37°C incubator.
4.稳转细胞株的建立4. Establishment of Stably Transduced Cell Lines
将目的基因通过磷酸钙法制备成携带外源目的基因的retrovirus(逆转录病毒)或 lentivirus(慢病毒)。得到病毒后，用Retrovirus或Lentivirus病毒上清液感染目的细胞，以2.5x 105/6cm培养皿的密度传代待感染细胞，24小时后，于4℃冰箱取出已过滤的病毒上清液，加入适量体积的4mg/ml polybrane液体(1：1000稀释，终浓度4μg/ml)，混匀，吸取适量病毒液至待感染细胞的培养基中，37℃培养箱培养。24小时后更换新鲜病毒液，继续感染细胞。感染结束后24～48小时，对于携带表达抗生素基因的病毒，加入相应的抗生素进行阳性克隆的筛选(Puromycin或G418，不同的细胞用不同的浓度进行筛选)，筛选5-7天；对于携带表达荧光标记基因的病毒，则用相应的荧光标记进行筛选(copGFP或DsRed2)。收集病毒感染且筛选过的细胞的总RNA和总蛋白，分别进行real-time RT-PCR和western blot检测这些细胞中目的基因转录和翻译水平的表达情况，以鉴定过表达外源基因或者shRNA沉默内源基因的稳定细胞株的建立。The target gene is prepared into retrovirus (retrovirus) or lentivirus (lentivirus) carrying exogenous target gene by calcium phosphate method. After obtaining the virus, infect the target cells with Retrovirus or Lentivirus virus supernatant, and pass the cells to be infected at a density of 2.5x 105/6cm culture dish. Volume of 4mg/ml polybrane liquid (1:1000 dilution, final concentration 4μg/ml), mix well, pipette an appropriate amount of virus liquid into the medium of the cells to be infected, and cultivate in a 37°C incubator. After 24 hours, replace with fresh virus solution and continue to infect cells. 24 to 48 hours after the infection, for viruses carrying antibiotic genes, add corresponding antibiotics to screen for positive clones (Puromycin or G418, different cells are screened with different concentrations), and screen for 5-7 days; Viruses with fluorescently labeled genes were screened with corresponding fluorescent markers (copGFP or DsRed2). Collect total RNA and total protein of virus-infected and screened cells, and perform real-time RT-PCR and western blot to detect the expression of target gene transcription and translation levels in these cells to identify overexpressed exogenous genes or shRNA silencing Establishment of stable cell lines with endogenous genes.
据此通过转染相关质粒构建H82R pBabe-mCherry-GFP-LC3稳转细胞株，H446pBabe-mCherry-GFP-LC3稳转细胞株，H82R pLko.1-Tet-shGGPS1稳转细胞株，pLko.1-Tet-shRAB7A稳转细胞株。Accordingly, the H82R pBabe-mCherry-GFP-LC3 stable transfection cell line, H446pBabe-mCherry-GFP-LC3 stable cell line, H82R pLko.1-Tet-shGGPS1 stable cell line, pLko.1- Tet-shRAB7A stably transfected cell line.
5.抗体5. Antibodies
所用抗体如表1。Antibodies used are listed in Table 1.
表1Table 1
Figure PCTCN2021127538-appb-000001
Figure PCTCN2021127538-appb-000001
6.动物实验6. Animal experiments
本发明人根据影像学，判断为小细胞肺癌特征后，利用穿刺的组织建立人源来源的小细胞肺癌肿瘤模型(PDX)，如果肿瘤在小鼠接种后成功长起来，并可以连续传代，可认为该PDX建立成功。成功建立20个SCLC PDX模型。动物实验流程：提前1周订购3-4周龄的裸鼠，使其在动物房适应1周。将需要接种的细胞扩增到大致的量(一般为5个10cm)，消化成单细胞后计数。按照每只裸鼠1×10 6的细胞(细胞量根据不同的细胞种类决定)，每组6只小鼠的量取对应体积的细胞悬液在新的离心管后离心，每只小鼠对应100μL的量用灭菌1×PBS重悬。用1mL的注射器将细胞悬液通过皮下注射到裸鼠的背部两侧或者前肢的腋下。如果是组织的话，接种组织块大小约3x3mm至小鼠背部皮下。1周之后观察是否长出肿瘤，随后开始每2天用游标卡尺记录肿瘤的大小(肿瘤体积＝长×宽×宽/2)。直到肿瘤体积达到2000mm 3或小鼠死亡结束记录，取下并称取小鼠皮下肿瘤重量，固定过夜后进行石蜡包埋、切片和H&E染色。 After judging the characteristics of small cell lung cancer based on imaging studies, the inventors established a human-derived small cell lung cancer tumor model (PDX) using the punctured tissue. The PDX is considered to be established successfully. Successfully built 20 SCLC PDX models. Animal experiment procedure: Nude mice aged 3-4 weeks were ordered 1 week in advance and allowed to adapt to the animal room for 1 week. The cells to be seeded were expanded to an approximate amount (usually 5 cells of 10 cm), digested into single cells and counted. According to 1×10 6 cells of each nude mouse (the amount of cells is determined according to different cell types), the corresponding volume of cell suspension was taken from each group of 6 mice and centrifuged in a new centrifuge tube. A 100 μL volume was resuspended in sterile 1×PBS. Using a 1 mL syringe, the cell suspension was subcutaneously injected into the back sides of the nude mice or the armpits of the forelimbs. In the case of tissue, inoculate a tissue block size of approximately 3x3 mm subcutaneously on the back of the mouse. After 1 week, it was observed whether the tumor grew, and then the size of the tumor was recorded every 2 days with a vernier caliper (tumor volume=length×width×width/2). Until the tumor volume reached 2000mm3 or the mice died, the mice were removed and the subcutaneous tumor weight was weighed. After being fixed overnight, paraffin embedding, sectioning and H&E staining were performed.
7.免疫组化7. Immunohistochemistry
采用免疫组织化学LSAB法(Labelled StreptAvidin-Biotin，抗生物素蛋白链菌素-生物素标记法)检测组织标本中目的蛋白的表达情况。Immunohistochemical LSAB method (Labelled StreptAvidin-Biotin, streptavidin-biotin labeling method) was used to detect the expression of target protein in tissue samples.
8.蛋白的收集和检测8. Collection and Detection of Proteins
样品的准备：将贴壁培养的细胞株弃去培养液(悬浮细胞离心去上清)，PBS漂洗两次，尽量吸尽残留的PBS液，每瓶加100-200μl 1×SDS蛋白裂解液，用细胞刮匙轻轻把细胞刮下，收集到1.5ml的EP管中，待裂解充分后，用1ml注射器将粘稠物上下吹打，至裂解液变成悬滴状为止，置-20℃冰箱保存备用；BCA(标准曲线)蛋白定量。Western Blot检测目的蛋白。细胞膜蛋白与细胞浆蛋白提取，使用碧云天(P0033)试剂盒抽提。Sample preparation: Discard the culture medium of the adherent cultured cell lines (suspend the cells by centrifugation to remove the supernatant), rinse with PBS twice, and try to absorb the remaining PBS solution, add 100-200 μl 1×SDS protein lysis buffer to each bottle, Gently scrape the cells with a cell scraper and collect them into a 1.5ml EP tube. After the lysis is complete, use a 1ml syringe to pipette the viscous material up and down until the lysate turns into a hanging drop. Save for later use; BCA (standard curve) protein quantification. The target protein was detected by Western Blot. Cell membrane protein and cytoplasmic protein were extracted using Biyuntian (P0033) kit.
9.药物处理9. Medication
E/P药物：在建立化疗耐药模型时，等肿瘤到给药体积，给予小鼠E/P药物处理，按照1周1个疗程计算，根据小鼠体重按照10毫克/千克/天第1，2，3天给予依托泊苷(VP16)，按6毫克/千克/天在第1天给予顺铂(CDDP)，配好药用生理盐水稀释，最终注射体积为0.2mL/只小鼠，腹腔注射。依托泊苷购买自sigma公司，货号E1383。顺铂购买自sigma公司，货号P4394。用DMSO(二甲基亚枫)配制成贮备浓度。依托泊苷为30mg/ml，顺铂为1.5mg/ml。E/P drugs: When establishing a chemotherapy resistance model, when the tumor reaches the dosed volume, the mice are treated with E/P drugs, calculated as a course of 1 week, and 10 mg/kg/day according to the body weight of the mice. , Etoposide (VP16) was administered on days 2 and 3, and cisplatin (CDDP) was administered on day 1 at 6 mg/kg/day, diluted with medicinal saline, and the final injection volume was 0.2 mL/mouse, Intraperitoneal injection. Etoposide was purchased from Sigma, Cat. No. E1383. Cisplatin was purchased from Sigma, Cat. No. P4394. Stock concentrations were formulated with DMSO (dimethyl sulfoxide). Etoposide is 30 mg/ml and cisplatin is 1.5 mg/ml.
Statin药物：等肿瘤到给药体积，给予小鼠Statin药物处理，每天给药，根据小鼠体重按照50毫克/千克/天通过灌胃给予美伐他汀，辛伐他汀，匹伐他汀等药物处理。配好药用生理盐水稀释，最终注射体积为0.2mL/只小鼠。美伐他汀购买自MCE公司，货号HY-17408。辛伐他汀购买自MCE公司，货号HY-17502，匹伐他汀购买自MCE公司，货号HY-B0165A。Statin drug: When the tumor reaches the dosed volume, the mice are treated with Statin drug, administered daily, according to the body weight of the mice, 50 mg/kg/day of mevastatin, simvastatin, pitavastatin and other drugs are administered by gavage . It was diluted with medicinal saline, and the final injection volume was 0.2 mL/mouse. Mevastatin was purchased from MCE Company, catalog number HY-17408. Simvastatin was purchased from MCE Company, product number HY-17502, and pitavastatin was purchased from MCE Company, product number HY-B0165A.
药库筛选实验：待处理细胞按照2500细胞每孔铺装96孔板，每孔体积100微升。 24小时后，将待处理药物按照2X浓度配制好。按每孔100微升的体积将药物加入96孔板，终体积200微升。所用药库信息为MCE公司FDA认证的代谢类小分子抑制剂，货号HYCPK4390，HYCPK4391，HYCPK4392，HYCPK4393。72小时后，加入CTG试剂检测。根据读值分析药库筛选数据。Drug library screening experiment: The cells to be treated are plated in a 96-well plate according to 2500 cells per well, and the volume of each well is 100 microliters. After 24 hours, the drug to be treated is prepared according to the 2X concentration. The drug was added to the 96-well plate in a volume of 100 microliters per well, with a final volume of 200 microliters. The information of the drug library used is the metabolic small molecule inhibitor certified by the FDA of MCE Company, the product number is HYCPK4390, HYCPK4391, HYCPK4392, HYCPK4393. After 72 hours, CTG reagent was added for detection. Drug library screening data were analyzed based on reads.
10.激光共聚焦显微镜检测自噬流10. Detection of Autophagic Flux by Confocal Laser Microscopy
根据之前提到的质粒建立稳转细胞系H82R pBabe-mCherry-GFP-LC3，H446pBabe-mCherry-GFP-LC3和DMS114pBabe-mCherry-GFP-LC3，将细胞铺在12孔板中，提前已加入盖玻片。24h后:A.药物实验:分别加入DMSO对照组，5μm美伐他汀(MevaStatin)处理组，5μm MevaStatin和2μm GGPP处理组B.基因敲低实验：慢病毒感染细胞敲低RAB7A和GGPS1。3天处理后，细胞收集在显微镜下检测。在莱卡公司SP8WLL显微镜下63倍检测。实验原理是基于不同pH值稳定的绿色和红色荧光蛋白强弱显示。绿色荧光蛋白的荧光信号会在在溶酶体酸性条件下淬火(pH值低于5)，而mRFP荧光信号在酸性条件下没有显著变化。绿色和红色合在一起的图像，自噬溶酶体显示为黄色点状物(即RFP阳性和GFP阳性)，而自噬小体显示红色点状物(即RFP阳性和GFP阴性)。自噬流流畅时，自噬溶酶体正常形成，为黄色点状物，而自噬流障碍时，自噬小体正常形成，但不能与溶酶体结合，形成自噬溶酶体，所以为红色点状物。Stable transfection cell lines H82R pBabe-mCherry-GFP-LC3, H446pBabe-mCherry-GFP-LC3 and DMS114pBabe-mCherry-GFP-LC3 were established according to the previously mentioned plasmids, and the cells were plated in 12-well plates, and coverslips had been added in advance piece. 24h later: A. Drug experiment: Add DMSO control group, 5μm MevaStatin (MevaStatin) treatment group, 5μm MevaStatin and 2μm GGPP treatment group B. Gene knockdown experiment: lentivirus infected cells knock down RAB7A and GGPS1. 3 days After treatment, cells were collected and examined under a microscope. Detected at 63x under the Leica SP8WLL microscope. The experimental principle is based on the stable display of green and red fluorescent proteins at different pH values. The fluorescence signal of green fluorescent protein is quenched under acidic lysosomal conditions (pH below 5), whereas the fluorescence signal of mRFP does not change significantly under acidic conditions. Combined green and red images, autophagolysosomes are shown as yellow puncta (ie RFP positive and GFP positive), while autophagosomes show red puncta (ie RFP positive and GFP negative). When autophagic flow is smooth, autophagolysosomes are normally formed as yellow dots, but when autophagic flow is blocked, autophagosomes are normally formed, but cannot combine with lysosomes to form autophagolysosomes, so For red dots.
实施例1、小细胞肺癌小鼠模型的化疗给药流程Example 1. Chemotherapy administration process of small cell lung cancer mouse model
为了尽可能知道小细胞肺癌化疗耐药过程中的细胞变化，本发明人用四株化疗敏感细胞系(H82、H209、H526和H146)和两株化疗耐药细胞系(H446和DMS114)，按照1×10 6/只的细胞数目皮下注射到裸鼠背侧，等肿瘤体积长至约100-200mm 3，分成两组：对照组给予生理盐水处理(图1A)；给药组按照标准流程(SOP)给予依托泊苷和顺铂(E/P)化疗药物处理(图1B)。若化疗药物处理后，肿瘤明显消退，本发明人会暂停给药，等肿瘤重新恢复生长后，再继续给予化疗药物处理；重复此过程，直至化疗药物无法有效抑制肿瘤生长，最终建立化疗耐药的小细胞肺癌模型(图1A)。在此过程，若小鼠状态很差或者肿瘤体积超过2000mm 3，本发明人会将肿瘤接种至另一只小鼠，重复给药过程直至得到耐药肿瘤。 In order to know as much as possible the cellular changes in the chemoresistance process of small cell lung cancer, the inventors used four chemosensitive cell lines (H82, H209, H526 and H146) and two chemoresistant cell lines (H446 and DMS114), according to the The number of cells at 1×10 6 / mouse was subcutaneously injected into the back of nude mice, and the tumor volume grew to about 100-200 mm 3 , and divided into two groups: the control group was treated with normal saline (Fig. 1A); SOP) were treated with etoposide and cisplatin (E/P) chemotherapeutic drugs (Fig. 1B). If the tumor regresses significantly after treatment with chemotherapeutic drugs, the inventors will suspend the administration of the drug, and then continue to administer the chemotherapeutic drugs after the tumor resumes growth; this process is repeated until the chemotherapeutic drugs cannot effectively inhibit the tumor growth, and finally chemoresistance is established. small cell lung cancer model (Figure 1A). During this process, if the mice are in poor condition or the tumor volume exceeds 2000 mm 3 , the inventors will inoculate the tumor into another mouse, and repeat the administration process until drug-resistant tumors are obtained.
实施例2、小细胞肺癌化疗耐药肿瘤模型的建立Example 2. Establishment of chemotherapy-resistant tumor model of small cell lung cancer
如图所示(图2A)，通过连续的药物处理，相对于P1代肿瘤对E/P化疗药物的敏感，H82、H209、H526和H146在后续传代中逐渐呈现出对化疗药物的耐受。H82在传至P4代，H209在传至P5代，H526在传至P4代，H146在传至P3代时，化疗药物已经无法抑制药物处理组的肿瘤生长，由此认为已成功获取小细胞肺癌化疗耐药肿瘤(图2A)。As shown (Fig. 2A), by sequential drug treatment, H82, H209, H526 and H146 gradually exhibited resistance to chemotherapeutic drugs in subsequent passages, relative to the sensitivity of P1 generation tumors to E/P chemotherapeutics. When H82 was passed to the P4 generation, H209 was passed to the P5 generation, H526 was passed to the P4 generation, and H146 was passed to the P3 generation, the chemotherapeutic drugs could no longer inhibit the tumor growth in the drug-treated group. Therefore, it is considered that small cell lung cancer has been successfully obtained. Chemotherapy-resistant tumors (Figure 2A).
生存分析的结果显示，在给予化疗药物处理的情况下，接种H82、H209和H526肿瘤的小鼠生存期长，而接种耐药肿瘤的小鼠生存期短(图2B，C)。IHC检测显示敏感肿瘤在给药处理的情况下，凋亡发生标志物CC3和DNA损伤标志物H2AX表达上升，细胞凋亡和DNA损伤增加。而化疗耐药肿瘤在给予药物处理情况下，CC3和H2AX没有变化，没有诱导细胞凋亡和引起DNA损伤(图2D)。这提示化疗耐药肿瘤已经对化疗药物不响应，小细胞肺癌化疗耐药模型建立成功。The results of the survival analysis showed that the mice inoculated with H82, H209 and H526 tumors had longer survival in the presence of chemotherapeutic drug treatment, while the survival of mice inoculated with drug-resistant tumors was shorter (Fig. 2B,C). IHC detection showed that the expression of apoptosis marker CC3 and DNA damage marker H2AX increased, and apoptosis and DNA damage increased in sensitive tumors under drug treatment. In contrast, chemotherapy-resistant tumors showed no changes in CC3 and H2AX, and did not induce apoptosis and cause DNA damage (Fig. 2D). This suggests that chemotherapy-resistant tumors have not responded to chemotherapy drugs, and the chemotherapy-resistant model of small cell lung cancer was successfully established.
本发明人也在裸鼠上接种小细胞肺癌化疗耐药细胞系H446和DMS114，发现肿瘤初始就对化疗药物不响应(图2E)。生存分析显示，在给予化疗药物处理的情况下，接种H446和DMS114肿瘤的小鼠生存期短(图2F，G)。The inventors also inoculated the small cell lung cancer chemotherapy-resistant cell lines H446 and DMS114 in nude mice, and found that the tumors initially did not respond to chemotherapy drugs (Fig. 2E). Survival analysis showed that mice inoculated with H446 and DMS114 tumors had short survival in the presence of chemotherapeutic drug treatment (Fig. 2F,G).
实施例3、利用小细胞肺癌化疗耐药小鼠模型建立原代细胞株Example 3. Establishment of Primary Cell Lines Using Small Cell Lung Cancer Chemo-resistant Mouse Models
为了深入开展小细胞肺癌耐药研究，本发明人将耐药肿瘤进行原代培养，成功地构建原代小细胞肺癌化疗耐药细胞株H82R，H209R和H526R。本发明人发现，相对于H82细胞，H82R细胞呈现出对化疗药物的高度耐受(图3A，B)；而H209R和H526R细胞并没有呈现出比H209和H526细胞更显著的化疗耐受(图3C，D)。说明化疗耐药机制可能和肿瘤微环境相关，而化疗耐药细胞H82R在体内和体外都对化疗药物处理高度耐受，更加适合后续在细胞上进行药物筛选和机制研究。In order to carry out in-depth research on the drug resistance of small cell lung cancer, the inventors carried out primary culture of drug-resistant tumors, and successfully constructed primary small cell lung cancer chemotherapy-resistant cell lines H82R, H209R and H526R. The inventors found that, compared with H82 cells, H82R cells exhibited high resistance to chemotherapeutic drugs (Fig. 3A, B); while H209R and H526R cells did not exhibit more significant chemoresistance than H209 and H526 cells (Fig. 3C, D). It shows that the chemoresistance mechanism may be related to the tumor microenvironment, and the chemoresistance cell H82R is highly resistant to chemotherapeutic drug treatment in vivo and in vitro, which is more suitable for subsequent drug screening and mechanism research on cells.
对其它小细胞肺癌化疗耐药细胞系H446、DMS114和H196的药物响应检测，显示它们对化疗药物处理都是高度耐受(图3E)。这说明小细胞肺癌化疗耐药细胞系H446和DMS114在体内和体外都保持对化疗药物的高度耐受(图3E)。Drug response testing of other small cell lung cancer chemoresistant cell lines H446, DMS114 and H196 showed that they were all highly resistant to chemotherapeutic drug treatment (Figure 3E). This indicated that the SCLC chemoresistant cell lines H446 and DMS114 maintained high resistance to chemotherapeutic drugs both in vivo and in vitro (Figure 3E).
实施例4、利用代谢类药物库进行功能学筛选Example 4. Functional screening using metabolic drug library
以上的结果说明，H82和H82R适合作为配对的细胞系进行药库筛选，为了更快和安全地应用于临床，本发明人选取FDA认证的256个代谢相关的药物抑制剂，实验流程按照图中所示(图4A)。对这些药物分类，在人类疾病治疗方面，这些小分子抑制剂可以归类为代谢类疾病(22％)，肿瘤的治疗(15.2％)，心血管疾病(14.8％)，炎症/免疫的治疗(13.7％)，神经类疾病(12.3％)，感染(9.4％)，内分泌类疾病(3.2％)和一些其他疾病(9.4％)(图4B)。对于小分子抑制剂涉及到代谢分类，可以分为脂质类代谢，氨基酸/蛋白质类代谢，葡萄糖代谢，核酸代谢，辅酶/维生素代谢以及其他类代谢(图4C)。The above results show that H82 and H82R are suitable as paired cell lines for drug library screening. In order to apply them to the clinic faster and safely, the inventors selected 256 FDA-approved drug inhibitors related to metabolism. The experimental flow is as shown in the figure. shown (Figure 4A). For the classification of these drugs, in the treatment of human diseases, these small molecule inhibitors can be classified as metabolic diseases (22%), tumor treatment (15.2%), cardiovascular disease (14.8%), inflammation/immune treatment ( 13.7%), neurological diseases (12.3%), infections (9.4%), endocrine diseases (3.2%) and some other diseases (9.4%) (Fig. 4B). For small molecule inhibitors, it involves metabolic classification, which can be divided into lipid metabolism, amino acid/protein metabolism, glucose metabolism, nucleic acid metabolism, coenzyme/vitamin metabolism and other types of metabolism (Figure 4C).
实施例5、Statin抑制化疗耐药H82R细胞生长Example 5. Statin inhibits the growth of chemotherapy-resistant H82R cells
药库筛选的结果显示，相对于H82，H82R不仅对E/P化疗药物耐药，对很多其它药物都有一定耐药性(蓝点所示)(图5A)，但对他汀(Statin)类药物(包括AtorvStatin、PitvaStatin、LovaStatin、MevaStatin、SimvaStatin和FluaStatin)则非常敏感(图5A)。为 了确定Statin是否对H82R细胞有很强的抑制效应，本发明人检测了H82和H82R细胞在不同药物浓度下对六种Statin的效应(图5B，C)，结果显示这些Statin类药物都能剂量依赖性抑制H82R细胞生长。在持续给药处理下，相对于H82细胞，H82R对单独E/P处理耐药，细胞生长不受影响。但对Statin处理和Statin加E/P的联合处理敏感，细胞生长受到显著抑制(图5D)。这提示Statin可以抑制化疗耐药H82R细胞生长。The results of drug library screening showed that, compared with H82, H82R was not only resistant to E/P chemotherapeutics, but also to many other drugs (shown by blue dots) (Figure 5A), but also resistant to statins. Drugs, including AtorvStatin, PitvaStatin, LovaStatin, MevaStatin, SimvaStatin, and FluaStatin, were very sensitive (Figure 5A). In order to determine whether Statin has a strong inhibitory effect on H82R cells, the inventors examined the effects of H82 and H82R cells on six statins at different drug concentrations (Fig. 5B, C), and the results showed that these statins can be dosed Dependent inhibition of H82R cell growth. Compared with H82 cells, H82R was resistant to E/P treatment alone, and cell growth was not affected under continuous administration treatment. However, sensitive to Statin treatment and combined treatment of Statin plus E/P, cell growth was significantly inhibited (Fig. 5D). This suggests that Statin can inhibit the growth of chemotherapy-resistant H82R cells.
实施例6、Statin抑制多株小细胞肺癌化疗耐药细胞的生长Example 6. Statin inhibits the growth of chemotherapy-resistant cells in multiple small cell lung cancers
为确定Statin对于其它小细胞肺癌化疗耐药细胞株是否具有同样的抑制效果，本发明人对敏感细胞株(H209、H526、H146)和耐药细胞株(DMS114、H446、H196)分别给予E/P处理，MevaStatin处理和E/P加上MevaStatin联合处理(图6A)。In order to determine whether Statin has the same inhibitory effect on other small cell lung cancer chemotherapy-resistant cell lines, the inventors administered E/ P treatment, MevaStatin treatment and combined E/P plus MevaStatin treatment (Figure 6A).
结果显示，相对于敏感细胞株，化疗耐药细胞株对Statin和E/P加上MevaStatin联合处理非常敏感，细胞生长受到显著抑制(图6B)，而对E/P处理耐药，细胞生长不受影响(图6B)。这些结果表明，Statin可以抑制多株化疗耐药细胞生长。The results showed that, compared with the sensitive cell lines, the chemo-resistant cell lines were very sensitive to the combined treatment of Statin and E/P plus MevaStatin, and their cell growth was significantly inhibited (Figure 6B). affected (Fig. 6B). These results suggest that Statin can inhibit the growth of multiple chemoresistant cells.
实施例7、Statin抑制H82R移植瘤生长Example 7. Statin inhibits the growth of H82R xenograft tumor
为了确定Statin是否在体内抑制H82R细胞生长，本发明人使用H82R细胞皮下成瘤，然后分成4组：对照组，E/P处理组，MevaStatin处理组，E/P和MevaStatin联合处理组。结果显示，H82R肿瘤对E/P处理耐药，而MevaStatin处理和E/P加MevaStatin联合处理可以有效抑制H82R肿瘤的生长(图7A，B)。同时这些药物处理不会影响小鼠的体重(图7A)。IHC组化结果显示，MevaStatin处理组和E/P加MevaStatin联合处理组中的肿瘤CC3和H2AX会显著上升，提示药物处理引起细胞凋亡和DNA损伤增加。而肿瘤CC3和H2AX在E/P处理组没有明显变化，说明H82R肿瘤已经对E/P处理耐受(图7C，D)。在给予其它Statin(PitvaStatin和SimvaStatin)处理以及这些Statin加E/P联合处理下，H82R肿瘤生长同样会受到显著的抑制，这些药物处理不会影响小鼠体重(图7E)。这说明Statin类药物不仅在体外抑制化疗耐药细胞H82R的生长，在体内也能有效抑制化疗耐药肿瘤H82R的生长。To determine whether Statin inhibits the growth of H82R cells in vivo, the inventors used H82R cells to subcutaneously form tumors, and then divided into 4 groups: control group, E/P treatment group, MevaStatin treatment group, E/P and MevaStatin combined treatment group. The results showed that H82R tumors were resistant to E/P treatment, while MevaStatin treatment and combined E/P plus MevaStatin treatment could effectively inhibit the growth of H82R tumors (Fig. 7A, B). At the same time these drug treatments did not affect the body weight of the mice (Fig. 7A). IHC histochemical results showed that tumor CC3 and H2AX were significantly increased in the MevaStatin-treated group and the E/P plus MevaStatin combined treatment group, suggesting that drug treatment caused increased apoptosis and DNA damage. However, tumor CC3 and H2AX did not change significantly in the E/P treatment group, indicating that the H82R tumor had become resistant to E/P treatment (Fig. 7C, D). H82R tumor growth was also significantly inhibited under treatment with other Statins (PitvaStatin and SimvaStatin) and in combination with these statins plus E/P, and these drug treatments did not affect mouse body weight (Fig. 7E). This indicates that statins not only inhibit the growth of chemotherapy-resistant H82R cells in vitro, but also effectively inhibit the growth of chemotherapy-resistant tumor H82R in vivo.
实施例8、GGPP逆转Statin对H82R细胞的抑制效应Example 8. GGPP reverses the inhibitory effect of Statin on H82R cells
小分子药物抑制性经常不够特异，为了明确具体哪个MVA途径下游代谢物对耐药细胞有着重要功能和作用。本发明人在给予Statin处理的情况下，同时回补MVA途径的下游代谢物。结果显示，除了MVA可以逆转Statin对H82R细胞的抑制效应，GGPP和GGOH也可以逆转Statin对H82R细胞的抑制效应。其中GGOH在细胞内不存在，但拥有和GGPP一样的基团，发挥蛋白香叶基香叶基化作用(图8A)。The inhibition of small molecule drugs is often not specific enough, in order to clarify which specific downstream metabolites of the MVA pathway have important functions and effects on drug-resistant cells. The present inventors supplemented the downstream metabolites of the MVA pathway at the same time in the case of administration of Statin. The results showed that in addition to MVA could reverse the inhibitory effect of Statin on H82R cells, GGPP and GGOH could also reverse the inhibitory effect of Statin on H82R cells. Among them, GGOH does not exist in cells, but has the same group as GGPP, and plays the role of protein geranylgeranylation (Fig. 8A).
为了进一步确定其它代谢物是否可以逆转Statin对H82R细胞的抑制效应，在给 予Statin处理的情况下，同时回补不同浓度的MVA途径的下游代谢物。结果显示，只有MVA、GGPP和GGOH可以逆转Statin对H82R细胞的抑制效应(图8B)。这提示GGPP是H82R细胞生长所必需的代谢物质。To further determine whether other metabolites could reverse the inhibitory effect of Statin on H82R cells, different concentrations of downstream metabolites of the MVA pathway were simultaneously supplemented in the presence of Statin treatment. The results showed that only MVA, GGPP and GGOH could reverse the inhibitory effect of Statin on H82R cells (Fig. 8B). This suggests that GGPP is an essential metabolite for H82R cell growth.
实施例9、Statin抑制H82R细胞内GGPP产生进而影响RAB7A的膜定位Example 9. Statin inhibits the production of GGPP in H82R cells and affects the membrane localization of RAB7A
MVA下游产物GGPP可以逆转Statin对耐药细胞的抑制效应，后续本发明人想研究GGPP发挥该作用的机制。GGPP主要通过蛋白翻译后的香叶基香叶基化修饰小G蛋白(Small GTP-ase)，使其结合于膜上，进一步被GTP酶磷酸化发挥功能。为了明确哪些小G蛋白被GGPP修饰从而在耐药细胞发挥功能，本发明人构建shRNA文库对H82R细胞进行目的基因敲低，该文库选取细胞中重要的小G蛋白(RHOA、RHOB、RAP1A、RAP1B、CDC42)，以及MVA通路中关键的代谢酶基因。同时根据RNA-seq结果(图9A)，选取在耐药细胞中有表达的RAB基因。结果显示敲低GGPS1和RAB7A是抑制化疗耐药细胞增殖最明显的两个基因(图9B)。而相对于H82R细胞，在H82细胞中敲低这两个基因则没有那么显著的生长抑制(图9C)。GGPP, the downstream product of MVA, can reverse the inhibitory effect of Statin on drug-resistant cells, and the inventors want to study the mechanism by which GGPP exerts this effect. GGPP mainly modifies small G protein (Small GTP-ase) through geranylgeranylation after protein translation, so that it binds to the membrane and is further phosphorylated by GTPase to function. In order to clarify which small G proteins are modified by GGPP to function in drug-resistant cells, the inventors constructed a shRNA library to knock down the target gene in H82R cells. The library selected important small G proteins (RHOA, RHOB, RAP1A, RAP1B) in cells , CDC42), and key metabolic enzyme genes in the MVA pathway. At the same time, according to the RNA-seq results (Fig. 9A), the RAB genes expressed in the drug-resistant cells were selected. The results showed that knockdown of GGPS1 and RAB7A were the two most obvious genes that inhibited the proliferation of chemoresistant cells (Fig. 9B). However, knockdown of these two genes in H82 cells did not result in as significant growth inhibition relative to H82R cells (Fig. 9C).
为了确定GGPP是通过香叶基香叶基化修饰RAB7A影响其在细胞膜上的定位，本发明人用细胞膜质分离实验来检测RAB7A在细胞膜上和细胞质里的表达情况。结果显示在正常情况下，RAB7A主要定位在H82R细胞膜上，少量表达于细胞质里。而在给予Statin处理后，会影响RAB7A的细胞膜膜定位，导致RAB7A主要堆积在细胞质里(图9D)。如果回补GGPP则可以回复RAB7A的细胞膜定位(图9D)。这些实验证明GGPP通过香叶基香叶基化修饰RAB7A使其结合于细胞膜上继而发挥功能。In order to determine that GGPP modifies RAB7A by geranylgeranylation and affects its localization on the cell membrane, the inventors used the cell membrane separation experiment to detect the expression of RAB7A on the cell membrane and in the cytoplasm. The results showed that under normal conditions, RAB7A was mainly localized on the H82R cell membrane, and a small amount was expressed in the cytoplasm. After treatment with Statin, the localization of RAB7A in the cell membrane was affected, resulting in the accumulation of RAB7A mainly in the cytoplasm (Fig. 9D). The membrane localization of RAB7A could be restored if GGPP was replenished (FIG. 9D). These experiments demonstrate that GGPP modifies RAB7A by geranylgeranylation to bind to the cell membrane and then function.
实施例10、敲低GGPS1或RAB7A引发化疗耐药细胞的自噬障碍Example 10. Knockdown of GGPS1 or RAB7A induces autophagy disorder in chemo-resistant cells
自噬溶酶体可以在细胞内清除损伤的蛋白和细胞器等，为细胞提供物质和能量。在整个过程中，细胞将待降解物吞噬到自噬小体，直到最后自噬溶酶体形成并降解物质的过程称为自噬流(Autophagic flux)。已有研究表明，自噬小体和溶酶体的结合需要RAB7A帮助形成自噬溶酶体。若抑制RAB7A功能会影响自噬溶酶体的形成，引起细胞自噬流障碍。为了确定H82R细胞中自噬流的状况，本发明人拟检测P62和LC3B的蛋白表达水平。其中LC3B指示细胞内自噬小体的形成。P62具有底物的特异性，它连接LC3B与待降解的泛素化蛋白进入到自噬小体后，再与溶酶体融合形成自噬溶酶体从而被降解，可以用来指示自噬流是否正常。自噬流正常的话，自噬小体增加，P62被降解，其表达降低。如果自噬溶酶体的形成受影响，引起自噬流障碍，会出现LC3B上升，而P62无法被降解，其表达水平不变。结果显示，Statin处理后，相对于H82细胞，LC3B在H82R中显著升高，但P62没有变化。这提示，Statin会引起细胞自噬流障碍。而回补GGPP则会回复LC3B和P62的蛋白表达水平。这提示GGPP会逆转 Statin引起的细胞自噬流障碍(图10A)。同时本发明人发现Statin处理后，细胞内PARP和CC3显著升高，细胞凋亡增多。而回补GGPP可以阻断细胞凋亡的发生(图10B)。Autophagy lysosomes can remove damaged proteins and organelles in cells and provide cells with substances and energy. During the whole process, cells phagocytose the degraded substances into autophagosomes, and the process of forming and degrading substances in autophagolysosomes is called autophagic flux. Studies have shown that the association of autophagosomes and lysosomes requires RAB7A to help form autophagolysosomes. Inhibition of RAB7A function will affect the formation of autophagolysosomes and cause autophagic flow disorders. In order to determine the status of autophagic flux in H82R cells, the inventors intend to detect the protein expression levels of P62 and LC3B. where LC3B indicates the formation of intracellular autophagosomes. P62 has substrate specificity. It connects LC3B and the ubiquitinated protein to be degraded into autophagosomes, and then fuses with lysosomes to form autophagolysosomes to be degraded, which can be used to indicate autophagic flow. Is it normal. When autophagic flux is normal, autophagosomes are increased, P62 is degraded, and its expression is decreased. If autophagolysosome formation is affected and autophagic flux is blocked, LC3B will increase, while P62 cannot be degraded and its expression level remains unchanged. The results showed that after Statin treatment, LC3B was significantly elevated in H82R relative to H82 cells, but there was no change in P62. This suggests that Statin causes autophagic flow disturbance. GGPP supplementation restored the protein expression levels of LC3B and P62. This suggested that GGPP reversed the Statin-induced impairment of autophagic flux (Fig. 10A). At the same time, the inventors found that after treatment with Statin, PARP and CC3 in cells were significantly increased, and cell apoptosis was increased. Apoptosis of GGPP could block the occurrence of apoptosis (Fig. 10B).
在其他小细胞肺癌细胞中给予Statin处理后，相对于化疗敏感细胞株(H209、H526)，化疗耐药细胞株(H446、DMS114)中LC3B显著上升，P62没有明显变化。这提示Statin会影响耐药细胞株中自噬溶酶体的形成，引起自噬流障碍(图10C)。Compared with chemotherapy-sensitive cell lines (H209, H526), LC3B was significantly increased in chemotherapy-resistant cell lines (H446, DMS114), and P62 was not significantly changed after Statin treatment in other small cell lung cancer cells. This suggested that Statin could affect the formation of autophagolysosomes in drug-resistant cell lines, causing autophagic flux barriers (Fig. 10C).
为了确定RAB7A是影响自噬溶酶体形成的关键。本发明人在H82R中敲低GGPS1或RAB7A，之后检测P62和LC3B的表达。结果显示敲低GGPS1或RAB7A，细胞内LC3B显著上升，P62没有明显变化。这提示GGPS1和RAB7A是保障化疗耐药细胞自噬流顺畅的关键蛋白(图10D)。同样本发明人发现敲低GGPS1或RAB7A会诱导细胞凋亡发生(图10E)。HMGCR作为MVA途径的起始基因，细胞内敲低HMGCR，也会引起LC3B和CC3显著上升(图10F)。To determine that RAB7A is critical for the formation of autophagic lysosomes. The inventors knocked down GGPS1 or RAB7A in H82R, and then detected the expression of P62 and LC3B. The results showed that knockdown of GGPS1 or RAB7A significantly increased intracellular LC3B, but no significant change in P62. This suggests that GGPS1 and RAB7A are key proteins that ensure smooth autophagic flow in chemotherapy-resistant cells (Fig. 10D). The inventors also found that knockdown of GGPS1 or RAB7A induced apoptosis (Fig. 10E). HMGCR, as the initiation gene of the MVA pathway, knockdown of HMGCR in cells also caused a significant increase in LC3B and CC3 (Fig. 10F).
在化疗耐药细胞株H446中敲低GGPS1或RAB7A会抑制细胞增殖。同时细胞内LC3B会显著上升，而P62没有明显变化(图10G，H)。Knockdown of GGPS1 or RAB7A in chemoresistant cell line H446 inhibited cell proliferation. At the same time, intracellular LC3B was significantly increased, while P62 did not change significantly (Fig. 10G, H).
在体内H82R肿瘤上，给予Statin处理或Statin加E/P处理，同样会引起LC3B显著上升，而P62没有明显变化(图10I)。这些结果提示，Statin通过抑制GGPP-RAB7A-Autophagic途径引发化疗耐药细胞的自噬流障碍。On H82R tumors in vivo, Statin treatment or Statin plus E/P treatment also caused a significant increase in LC3B, but no significant change in P62 (Fig. 10I). These results suggest that Statin induces autophagic flux impairment in chemo-resistant cells by inhibiting the GGPP-RAB7A-Autophagic pathway.
实施例11、双荧光系统示踪化疗耐药细胞的自噬流Example 11. Dual Fluorescence System Tracking Autophagy Flux of Chemo-resistant Cells
为了直接观察抑制GGPP的产生会引起化疗耐药细胞的自噬流障碍，本发明人用荧光标记示踪的方法检测自噬的发生过程。在建立H82R pBabe-mCherry-GFP-LC3和H446pBabe-mCherry-GFP-LC3稳转细胞株后，本发明人给予细胞Statin处理，进一步通过免疫荧光来观察细胞内的自噬流状况。结果显示在对照组细胞中，指示自噬流正常的RFP+GFP-puncta处于较高水平，指示自噬流障碍的RFP+GFP+puncta处于低水平。而在Statin处理组中，RFP+GFP-puncta变低，指示自噬流障碍的RFP+GFP+puncta显著上升。这时回补GGPP可以使RFP+GFP-puncta变高，RFP+GFP+puncta降低，细胞自噬流回复正常。这提示Statin处理会引起化疗耐药细胞的自噬流障碍，而补充GGPP可以使细胞自噬流回复正常(图11A，B)。In order to directly observe that inhibiting the production of GGPP will cause the autophagy flow barrier of chemo-resistant cells, the inventors used the method of fluorescent labeling to detect the occurrence process of autophagy. After establishing H82R pBabe-mCherry-GFP-LC3 and H446pBabe-mCherry-GFP-LC3 stably transfected cell lines, the inventors treated the cells with Statin, and further observed the intracellular autophagic flow by immunofluorescence. The results showed that in the cells of the control group, the level of RFP+GFP-puncta indicating normal autophagic flux was high, and the level of RFP+GFP+puncta indicating impaired autophagic flux was low. Whereas, in the Statin-treated group, RFP+GFP-puncta decreased, and RFP+GFP+puncta, which is indicative of autophagic flow disturbance, was significantly increased. At this time, replenishing GGPP can make RFP+GFP-puncta increase, RFP+GFP+puncta decrease, and the autophagic flow returns to normal. This suggested that Statin treatment caused a disturbance of autophagic flux in chemo-resistant cells, whereas supplementation of GGPP could normalize autophagic flux (Fig. 11A,B).
在H446细胞敲低GGPS1或RAB7A，同样发现RFP+GFP-puncta变低，而指示细胞自噬流障碍的RFP+GFP+puncta上升(图11C)。In H446 cells knockdown of GGPS1 or RAB7A, RFP+GFP-puncta was also found to be low, while RFP+GFP+puncta, which indicates a disorder of autophagic flow, was increased (FIG. 11C).
这些结果表明，Statin会抑制MVA途径的GGPP产生，使RAB7A无法结合到细胞膜上，阻碍自噬溶酶体形成，引起自噬流障碍，诱导细胞凋亡，而外源补充GGPP则可以回复细胞生长。These results suggest that Statin inhibits the production of GGPP in the MVA pathway, making RAB7A unable to bind to the cell membrane, hindering the formation of autophagic lysosomes, causing autophagic flow barriers and inducing apoptosis, while exogenous supplementation of GGPP can restore cell growth .
实施例12、敲低GGPS1或RAB7A抑制化疗耐药肿瘤生长Example 12. Knockdown of GGPS1 or RAB7A inhibits the growth of chemotherapy-resistant tumors
为了确定敲低GGPS1或RAB7A会抑制小细胞肺癌化疗耐药细胞移植瘤生长。在建立H82R pLko.1-Tet-shGGPS1和pLko.1-Tet-shRAB7A稳转细胞后，将细胞在裸鼠皮下成瘤，分成对照组处理和实验组处理：其中对照组小鼠喂正常的水；实验组小鼠喂含有Dox(Doxycycline)的水。结果显示在细胞敲低GGPS1的情况下，肿瘤生长受到显著抑制(图12A)，甚至有40％比例的肿瘤完全被抑制，没有生长(图12A)。Western Blot(WB)结果显示肿瘤敲低GGPS1后，LC3B显著上升，而P62没有明显变化，提示在体内肿瘤敲低GGPS1会引起自噬流障碍(图12B)。IHC结果显示肿瘤在敲低GGPS1后，CC3和H2AX水平显著上升，肿瘤中凋亡发生和DNA损伤显著增加(图12C)。To determine whether knockdown of GGPS1 or RAB7A inhibits the growth of small cell lung cancer chemotherapy-resistant cell xenografts. After the establishment of H82R pLko.1-Tet-shGGPS1 and pLko.1-Tet-shRAB7A stably transfected cells, the cells were subcutaneously formed in nude mice and divided into control group treatment and experimental group treatment: the control group mice were fed with normal water ; The mice in the experimental group were fed water containing Dox (Doxycycline). The results showed that tumor growth was significantly inhibited in the case of cells knocking down GGPS1 (Figure 12A), and even 40% of the tumors were completely inhibited and did not grow (Figure 12A). Western Blot (WB) results showed that after tumor knockdown of GGPS1, LC3B was significantly increased, while P62 did not change significantly, suggesting that tumor knockdown of GGPS1 in vivo caused autophagy flow disorder (Figure 12B). IHC results showed that after GGPS1 knockdown in tumors, the levels of CC3 and H2AX were significantly increased, and apoptosis and DNA damage were significantly increased in tumors ( FIG. 12C ).
同样在细胞敲低RAB7A的情况下，肿瘤生长受到抑制(图12D)。WB结果显示显示肿瘤敲低RAB7A后，LC3B显著上升，而P62没有明显变化，提示在体内肿瘤敲低RAB7A会引起自噬流障碍(图12E)。免疫组化结果显示肿瘤在敲低RAB7A后，CC3和H2AX水平显著上升，肿瘤中凋亡发生和DNA损伤显著增加(图12F)。同时本发明人注意到敲低GGPS1比敲低RAB7A对于肿瘤的抑制效果更为明显，提示这是因为GGPS1对于细胞的功能非常重要，受影响的小G蛋白众多，而不仅仅是RAB7A。Also in the case of cells knocking down RAB7A, tumor growth was inhibited (FIG. 12D). WB results showed that after tumor knockdown of RAB7A, LC3B was significantly increased, while P62 did not change significantly, suggesting that tumor knockdown of RAB7A in vivo caused autophagic flow impairment (Figure 12E). Immunohistochemical results showed that after knockdown of RAB7A in tumors, the levels of CC3 and H2AX were significantly increased, and apoptosis and DNA damage were significantly increased in tumors (Fig. 12F). At the same time, the inventors noticed that knockdown of GGPS1 has a more obvious inhibitory effect on tumors than knockdown of RAB7A, suggesting that this is because GGPS1 is very important for cell function, and many small G proteins are affected, not just RAB7A.
实施例13、Statin抑制高表达GGPS1的小细胞肺癌化疗耐药PDX肿瘤生长Example 13. Statin inhibits the growth of chemotherapy-resistant PDX tumors in small cell lung cancer with high expression of GGPS1
为了确定Statin对于小细胞肺癌PDX肿瘤的治疗效果，本发明人建立了小细胞肺癌化疗耐药PDX肿瘤小鼠模型(图13A)。在建立的SCLC PDX ZS7和ZS4(ZS7和ZS4指模型代号)中，本发明人发现接种的肿瘤初始对于E/P处理敏感，后续在体内连续给予化疗药物处理下，肿瘤逐渐耐药，直至获得小细胞肺癌化疗耐药肿瘤PDX ZS7R和ZS4R(图13A，B)(ZS7R和ZS4R指耐药模型代号)。肿瘤的生长曲线和生存分析显示相对于PDX肿瘤ZS7和ZS4，ZS7R和ZS4R已经对E/P化疗药物处理耐受(图13A，B)。In order to determine the therapeutic effect of Statin on small cell lung cancer PDX tumors, the inventors established a small cell lung cancer chemotherapy-resistant PDX tumor mouse model ( FIG. 13A ). In the established SCLC PDX ZS7 and ZS4 (ZS7 and ZS4 refer to the model code), the inventors found that the inoculated tumors were initially sensitive to E/P treatment, and subsequently treated with chemotherapy drugs in vivo, the tumors gradually became resistant until the Small cell lung cancer chemotherapy-resistant tumors PDX ZS7R and ZS4R (Fig. 13A, B) (ZS7R and ZS4R refer to drug resistance model code). Growth curves and survival analysis of tumors showed that ZS7R and ZS4R were already resistant to E/P chemotherapeutic drug treatment relative to PDX tumors ZS7 and ZS4 (Figure 13A,B).
通过WB检测ZS7、ZS7R和ZS4、ZS4R中MVA相关基因的表达，结果显示相对于敏感肿瘤，ZS7R中MVA途径的FDPS和GGPS1高表达，而FDFT1和SQLE低表达。ZS4R中MVA途径的FDPS和GGPS1低表达，而FDFT1和SQLE高表达，说明MVA途径在ZS7A(化疗药物敏感模型代号)更多的合成GGPP，而ZS4R更多的合成胆固醇(图13C)。这提示相对于ZS4R，ZS7R更适合给予Statin治疗。体内的结果证明Statin和Statin加E/P联合处理组显著抑制ZS7R肿瘤的生长，而对ZS4R肿瘤没有抑制效应。ZS7R和ZS4R肿瘤都对E/P处理耐药(图13D)。其中也可见，Statin加E/P联合处理组的效果显著优于Statin单处理或E/P单处理组。The expression of MVA-related genes in ZS7, ZS7R and ZS4, ZS4R was detected by WB. The results showed that FDPS and GGPS1 of the MVA pathway in ZS7R were highly expressed, while FDFT1 and SQLE were low expressed in ZS7R compared with sensitive tumors. The MVA pathway in ZS4R has low expression of FDPS and GGPS1, but high expression of FDFT1 and SQLE, indicating that the MVA pathway is more GGPP synthesized in ZS7A (chemotherapy drug-sensitive model code), while ZS4R is more synthesized in cholesterol (FIG. 13C). This suggests that ZS7R is more suitable for Statin treatment than ZS4R. The in vivo results demonstrated that the combined treatment of Statin and Statin plus E/P significantly inhibited the growth of ZS7R tumors, but had no inhibitory effect on ZS4R tumors. Both ZS7R and ZS4R tumors were resistant to E/P treatment (Figure 13D). It can also be seen that the effect of Statin plus E/P combined treatment group was significantly better than that of Statin single treatment or E/P single treatment group.
这说明小细胞肺癌化疗耐药过程中，若肿瘤GGPS1水平升高可以用来指示使用Statin靶向治疗。This indicates that in the process of chemotherapy resistance in small cell lung cancer, if the level of tumor GGPS1 is elevated, it can be used to indicate the use of Statin-targeted therapy.
实施例14、Statin抑制临床上高表达GGPS1的小细胞肺癌化疗耐药PDX肿瘤生 长Example 14. Statin inhibits the growth of chemotherapy-resistant PDX tumors in small cell lung cancer with high GGPS1 expression
为了确定Statin可以用于临床治疗。本发明人与医院合作，获取对化疗药物响应不同的病人肿瘤组织，并将其建成小细胞肺癌PDX模型。目前临床上根据病人对药物的响应可以分为四种情况：In order to determine that Statin can be used for clinical treatment. The inventors cooperated with hospitals to obtain tumor tissues from patients with different responses to chemotherapeutic drugs, and build them into a PDX model of small cell lung cancer. At present, clinically, according to the patient's response to the drug, it can be divided into four situations:
PD：疾病进展(progressive disease)，靶病灶最大径之和至少增加≥20％，或出现新病灶。SD：疾病稳定(stable disease)，靶病灶最大径之和缩小未达PR，或增大未达PD。PR：部分缓解(partial response)，靶病灶最大径之和减少≥30％。CR:完全缓解(complete response)，靶病灶完全消失。PD: Progressive disease, with an increase of at least ≥20% in the sum of the largest diameters of target lesions, or the appearance of new lesions. SD: stable disease, the sum of the largest diameters of target lesions does not reduce to PR, or the increase does not reach PD. PR: Partial response, with a reduction of ≥30% in the sum of the largest diameters of target lesions. CR: Complete response (complete response), the target lesions completely disappeared.
IHC和WB结果显示在10个小细胞肺癌PDX中，处于SD和PD阶段的小细胞肺癌病人GGPS1高表达(图14A，B)，其中在处于PD的小细胞肺癌病人中，GGPS1表达最高(图14A，B，C)。为了确定Statin对于处于PD阶段的小细胞肺癌病人有好的疗效，本发明人接种处于PD阶段的小细胞肺癌PDX肿瘤SP9(肿瘤模型代号)，并分组给予不同药物处理。结果显示Statin和Statin加E/P处理显著抑制SP9的肿瘤生长。E/P处理也部分抑制肿瘤的生长，提示是肿瘤组织取材时，该病人尚未给予化疗药物治疗，存在一部分细胞处于不耐药的状态，所以联合处理会显著抑制肿瘤的生长(图14D，E)。免疫组化结果显示，肿瘤CC3和H2AX水平在Statin和Statin加E/P联合处理下显著上升，肿瘤中凋亡发生和DNA损伤显著增加(图14F)。IHC and WB results showed that in 10 small cell lung cancer PDXs, GGPS1 was highly expressed in small cell lung cancer patients in SD and PD stages (Fig. 14A, B), and GGPS1 expression was highest in PD small cell lung cancer patients (Fig. 14A, B). 14A, B, C). In order to confirm that Statin has a good curative effect on patients with small cell lung cancer in PD stage, the inventors inoculated SP9 (tumor model code) of small cell lung cancer in PD stage, and grouped them into different drug treatments. The results showed that Statin and Statin plus E/P treatment significantly inhibited SP9 tumor growth. E/P treatment also partially inhibited tumor growth, suggesting that when the tumor tissue was taken, the patient had not been treated with chemotherapy drugs, and some cells were in a state of non-resistance, so combined treatment would significantly inhibit tumor growth (Figure 14D, E ). Immunohistochemical results showed that tumor CC3 and H2AX levels were significantly increased under the combined treatment of Statin and Statin plus E/P, and apoptosis and DNA damage were significantly increased in tumors ( FIG. 14F ).
实施例15、TCGA数据库分析显示高表达GGPS1的小细胞肺癌患者预后更差Example 15. Analysis of TCGA database shows that patients with small cell lung cancer with high expression of GGPS1 have a worse prognosis
接下来通过TCGA数据库分析MVA途径相关基因表达水平和病人预后的相关性。结果显示高表达GGPS1的小细胞肺癌病人预后差(图15A)。而MVA途径其他基因(SQLE，FDFT1)与小细胞肺癌病人的预后没有相关性(图15B)。这说明高表达GGPS1的小细胞肺癌患者可能通过GGPP-RAB7A-Autophagic途径对化疗药物耐受，而MVA途径其它下游通路则不影响小细胞肺癌患者预后。Next, the correlation between MVA pathway-related gene expression levels and patient prognosis was analyzed through the TCGA database. The results showed that small cell lung cancer patients with high expression of GGPS1 had poor prognosis (Fig. 15A). However, other genes in the MVA pathway (SQLE, FDFT1) were not associated with the prognosis of small cell lung cancer patients (Fig. 15B). This indicates that patients with small cell lung cancer with high expression of GGPS1 may be resistant to chemotherapy drugs through the GGPP-RAB7A-Autophagic pathway, while other downstream pathways of the MVA pathway do not affect the prognosis of patients with small cell lung cancer.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (18)

  1. 一种筛选抑制化疗耐药小细胞肺癌的物质的方法，其特征在于，所述方法包括：A method for screening substances for inhibiting chemotherapy-resistant small cell lung cancer, characterized in that the method comprises:
    (1)将候选物质与含有GGPS1/RAB7A/自噬流信号通路的体系接触；(1) Contact the candidate substance with a system containing the GGPS1/RAB7A/autophagy flow signaling pathway;
    (2)筛选出调节GGPS1/RAB7A/自噬流信号通路的物质，所述物质是对于抑制化疗耐药小细胞肺癌有用的物质；(2) Screening out a substance that regulates the GGPS1/RAB7A/autophagy flow signaling pathway, and the substance is a useful substance for inhibiting chemotherapy-resistant small cell lung cancer;
    其中，所述的调节包括：抑制GGPS1的表达或活性，抑制RAB7A的膜定位，抑制GGPS1代谢产物GGPP对RAB7A的修饰作用，或促进自噬流障碍。The regulation includes: inhibiting the expression or activity of GGPS1, inhibiting the membrane localization of RAB7A, inhibiting the modification effect of GGPS1 metabolite GGPP on RAB7A, or promoting autophagic flow disorder.
  2. 如权利要求1所述的方法，其特征在于，所述的GGPS1/RAB7A/自噬流信号通路被包含于甲羟戊酸途径中，或为甲羟戊酸途径的下游通路；或The method of claim 1, wherein the GGPS1/RAB7A/autophagy flow signaling pathway is included in the mevalonate pathway, or is a downstream pathway of the mevalonate pathway; or
    所述的GGPS1/RAB7A/自噬流信号通路包括：GGPS1蛋白，RAB7A蛋白；所述自噬流为自噬体与溶酶体相互融合引发的自噬流。The GGPS1/RAB7A/autophagic flux signaling pathway includes: GGPS1 protein, RAB7A protein; the autophagic flux is the autophagic flux triggered by the fusion of autophagosomes and lysosomes.
  3. 如权利要求1所述的方法，其特征在于，步骤(1)包括：向含有GGPS1/RAB7A/自噬流信号通路的体系中添加候选物质；和The method of claim 1, wherein step (1) comprises: adding candidate substances to the system containing the GGPS1/RAB7A/autophagy flow signaling pathway; and
    步骤(2)包括：检测GGPS1/RAB7A/自噬流信号通路中各蛋白或其编码基因的变化，并与对照组比较，其中所述的对照组是不添加所述候选物质的、含有GGPS1/RAB7A/自噬流信号通路的体系；Step (2) includes: detecting the changes of each protein or its encoding gene in the GGPS1/RAB7A/autophagy flow signaling pathway, and comparing it with a control group, wherein the control group does not add the candidate substance and contains GGPS1/ The system of RAB7A/autophagy flow signaling pathway;
    若候选物质抑制GGPS1的表达或活性，抑制RAB7A的膜定位，抑制GGPS1代谢产物GGPP对RAB7A的修饰作用，或促进自噬流障碍，则该候选物质是对于抑制化疗耐药小细胞肺癌有用的物质。If the candidate substance inhibits the expression or activity of GGPS1, inhibits the membrane localization of RAB7A, inhibits the modification of RAB7A by the GGPS1 metabolite GGPP, or promotes autophagy flow disorder, the candidate substance is a useful substance for inhibiting chemotherapy-resistant small cell lung cancer .
  4. 如权利要求1所述的方法，其特征在于，所述的含有GGPS1/RAB7A/自噬流信号通路的体系选自：细胞体系、亚细胞体系、组织体系或动物体系。The method of claim 1, wherein the system containing the GGPS1/RAB7A/autophagy flow signaling pathway is selected from the group consisting of: a cellular system, a subcellular system, a tissue system or an animal system.
  5. 如权利要求1所述的方法，其特征在于，所述的候选物质包括：针对GGPS1/RAB7A/自噬流信号通路、或其通路蛋白、或其上游或下游蛋白或基因设计的调控分子，CRISPR构建物，小分子化合物，来自化合物库的化合物。The method of claim 1, wherein the candidate substances comprise: regulatory molecules designed for GGPS1/RAB7A/autophagy flow signaling pathway, or its pathway proteins, or its upstream or downstream proteins or genes, CRISPR Constructs, small molecule compounds, compounds from compound libraries.
  6. GGPS1/RAB7A/自噬流信号通路的用途，用于筛选抑制化疗耐药小细胞肺癌的物质；较佳地，所述的GGPS1/RAB7A/自噬流信号通路被包含于甲羟戊酸途径中，或为甲羟戊酸途径的下游通路；或Use of GGPS1/RAB7A/autophagy flux signaling pathway for screening substances that inhibit chemotherapy-resistant small cell lung cancer; preferably, the GGPS1/RAB7A/autophagic flux signaling pathway is included in the mevalonate pathway , or a downstream pathway of the mevalonate pathway; or
    所述的GGPS1/RAB7A/自噬流信号通路包括：GGPS1蛋白，RAB7A蛋白；所述自噬流为自噬体与溶酶体相互融合引发的自噬流。The GGPS1/RAB7A/autophagic flux signaling pathway includes: GGPS1 protein, RAB7A protein; the autophagic flux is the autophagic flux triggered by the fusion of autophagosomes and lysosomes.
  7. 调节GGPS1/RAB7A/自噬流信号通路的调节剂的用途，用于制备抑制化疗耐药小细胞肺癌的药物组合物；其中，所述的调节剂包括选自下组：GGPS1的表达或活性抑制剂，RAB7A膜定位抑制剂，GGPS1代谢产物GGPP对RAB7A的修饰作用的抑制剂，或促进自噬流障碍的促进剂。Use of a regulator for regulating GGPS1/RAB7A/autophagy flow signaling pathway, for preparing a pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer; wherein, the regulator comprises a regulator selected from the group consisting of inhibiting the expression or activity of GGPS1 agents, inhibitors of RAB7A membrane localization, inhibitors of the modification of RAB7A by the GGPS1 metabolite GGPP, or promoters that promote disorders of autophagic flux.
  8. 如权利要求7所述的用途，其特征在于，所述GGPS1的表达或活性抑制剂包括：敲除或沉默GGPS1基因的试剂，抑制GGPS1蛋白活性的试剂；较佳地，包括：特异性干扰GGPS1基因表达的干扰分子，针对GGPS1基因的CRISPR基因编辑试剂、同源重组试剂或定点突变试剂，所述试剂将GGPS1进行功能丧失性突变；The use according to claim 7, wherein the GGPS1 expression or activity inhibitor comprises: a reagent for knocking out or silencing GGPS1 gene, a reagent for inhibiting the activity of GGPS1 protein; preferably, comprising: specifically interfering with GGPS1 Interfering molecules for gene expression, CRISPR gene editing reagents, homologous recombination reagents or site-directed mutagenesis reagents for the GGPS1 gene, the reagents mutate GGPS1 with loss-of-function;
    所述RAB7A膜定位抑制剂包括：敲除或沉默RAB7A基因的试剂，抑制RAB7A蛋白活性的试剂；较佳地，包括：特异性干扰RAB7A基因表达的干扰分子，针对RAB7A基因的CRISPR基因编辑试剂、同源重组试剂或定点突变试剂，所述试剂将RAB7A进行功能丧失性突变；The RAB7A membrane localization inhibitor includes: a reagent for knocking out or silencing the RAB7A gene, a reagent for inhibiting the activity of the RAB7A protein; preferably, it includes: an interfering molecule that specifically interferes with the expression of the RAB7A gene, a CRISPR gene editing reagent for the RAB7A gene, Homologous recombination reagents or site-directed mutagenesis reagents that mutate RAB7A loss-of-function;
    所述GGPS1代谢产物GGPP对RAB7A的修饰作用的抑制剂包括：抑制GGPS1合成GGPP的试剂；或The inhibitor of the modification effect of the GGPS1 metabolite GGPP on RAB7A includes: an agent that inhibits the synthesis of GGPP by GGPS1; or
    所述促进自噬流障碍的促进剂包括：GGPS1的表达或活性抑制剂，RAB7A膜定位抑制剂，GGPS1代谢产物GGPP对RAB7A的修饰作用的抑制剂。The promoters for promoting autophagic flow disorder include: inhibitors of the expression or activity of GGPS1, inhibitors of RAB7A membrane localization, and inhibitors of the modification effect of GGPS1 metabolite GGPP on RAB7A.
  9. 如权利要求8所述的用途，其特征在于，所述的敲除或沉默GGPS1基因的试剂为敲低GGPS1的shRNA质粒；用于敲低的功能序列为：CCTGAGCTAGTAGCCTTAGTA所述的敲除或沉默RAB7A基因的试剂为敲低RAB7A的shRNA质粒；用于敲低的功能序列为：GGCTAGTCACAATGCAGATAT。The use according to claim 8, wherein the reagent for knocking out or silencing GGPS1 gene is a shRNA plasmid for knocking down GGPS1; the functional sequence for knocking down is: the knockout or silencing RAB7A described in CCTGAGCTAGTAGCCTTAGTA The reagent for the gene is a shRNA plasmid for knocking down RAB7A; the functional sequence for knocking down is: GGCTAGTCACAATGCAGATAT.
  10. 他汀类药物、依托泊苷和顺铂的组合在制备抑制化疗耐药小细胞肺癌的药物组合物中的用途。Use of a combination of statins, etoposide and cisplatin in the preparation of a pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer.
  11. 一种抑制化疗耐药小细胞肺癌的药物组合物，其特征在于，包括他汀类药物、依托泊苷和顺铂。A pharmaceutical composition for inhibiting chemotherapy-resistant small cell lung cancer, comprising statins, etoposide and cisplatin.
  12. 一种抑制化疗耐药小细胞肺癌的药盒，其特征在于，其中包括权利要求11所 述的药物组合物；或其中包括容器，以及分别置于所述容器中的他汀类药物、依托泊苷和顺铂。A kit for inhibiting chemotherapy-resistant small cell lung cancer, comprising the pharmaceutical composition of claim 11; or a container, and statins and etoposide respectively placed in the container and cisplatin.
  13. 如权利要求10～12任一所述，其特征在于，所述他汀类药物包括选自：美伐他汀，辛伐他汀，匹伐他汀，阿托伐他汀，洛伐他汀，FluaStatin；或The statin drug according to any one of claims 10 to 12, wherein the statin is selected from the group consisting of mevastatin, simvastatin, pitavastatin, atorvastatin, lovastatin, and FluaStatin; or
    所述的化疗耐药小细胞肺癌为高表达GGPS1耐药性的小细胞肺癌。The chemotherapy-resistant small cell lung cancer is a small cell lung cancer with high expression of GGPS1 resistance.
  14. 如权利要求10～12任一所述，其特征在于，用于一个单位疗程时，所述的他汀类药物、依托泊苷和顺铂的用量比例为(35～105)：(3～9)：1；较佳地为(49～70)：(4.5～7.5)：1；或The method according to any one of claims 10 to 12, wherein when used for one unit course of treatment, the dosage ratio of the statins, etoposide and cisplatin is (35 to 105): (3 to 9) : 1; preferably (49-70): (4.5-7.5): 1; or
    所述顺铂、依托泊苷和所述他汀类药物的用药方法为：按1周1个疗程计算，根据个体体重，通过腹腔注射方式第1天给予0.5～10毫克/千克顺铂；第1～3天给予0.5～15毫克/千克/天依托泊苷；同时通过灌胃方式给予2～100毫克/千克/天的美伐他汀或辛伐他汀或匹伐他汀等药物处理；较佳地，当药物被置于药盒中时，所述用药方法被记载于所述药盒的使用说明书中。The medication methods of the cisplatin, etoposide and the statins are as follows: one course of treatment per week, according to individual body weight, 0.5-10 mg/kg of cisplatin is given by intraperitoneal injection on the first day; Administer 0.5-15 mg/kg/day etoposide for ~3 days; at the same time, administer 2-100 mg/kg/day mevastatin or simvastatin or pitavastatin by gavage; preferably, When the drug is placed in a kit, the method of administration is described in the instructions for use of the kit.
  15. GGPS1蛋白或其编码基因在制备诊断试剂中的用途，所述的诊断试剂用于对小细胞肺癌进行诊断或预后；较佳地，所述的诊断或预后包括：根据GGPS1蛋白的表达情况，判断其是否适用于进行他汀类药物的治疗方案；若GGPS1蛋白高表达，则适用该治疗方案。Use of GGPS1 protein or its encoding gene in preparing a diagnostic reagent for diagnosing or prognosing small cell lung cancer; preferably, the diagnosing or prognosing includes: judging the Whether it is suitable for the treatment of statins; if the GGPS1 protein is highly expressed, the treatment is applicable.
  16. 特异性识别GGPS1蛋白或其编码基因的试剂的用途，用于制备对小细胞肺癌进行诊断或预后的诊断试剂或诊断试剂盒；较佳地，所述的诊断或预后包括：根据GGPS1蛋白的表达情况，判断其是否适用于进行他汀类药物的治疗方案；若GGPS1蛋白高表达，则适用该治疗方案。Use of a reagent that specifically recognizes GGPS1 protein or its encoding gene, for preparing a diagnostic reagent or diagnostic kit for diagnosing or prognosing small cell lung cancer; preferably, the diagnosis or prognosis includes: according to the expression of GGPS1 protein If there is a high expression of GGPS1 protein, it is suitable for the treatment plan of statins.
  17. 如权利要求15或16所述，其特征在于，所述的治疗方案为他汀类药物、依托泊苷和顺铂联合用药的治疗方案。The method according to claim 15 or 16, wherein the treatment regimen is a treatment regimen of statins, etoposide and cisplatin in combination.
  18. 如权利要求15或16任一所述，其特征在于，所述的诊断试剂包括选自：特异性扩增GGPS1蛋白的编码基因的引物；特异性识别GGPS1蛋白的编码基因或其转录本的探针；或特异性抗GGPS1蛋白的抗体。The one according to claim 15 or 16, wherein the diagnostic reagent comprises a primer selected from the group consisting of: primers that specifically amplify the gene encoding GGPS1 protein; probes that specifically recognize the gene encoding GGPS1 protein or its transcript. needle; or an antibody specific for the GGPS1 protein.
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