CN103656683A - Application of pattern recognition receptors RIG-I in preparation of antitumor drug - Google Patents

Abstract

The invention relates to the field of biotechnology and medical diagnosis. According to the invention, molecule-pattern recognition receptors RIG-I with tumor expression correlation and having regulation effect on the growth of cancer cells are screened out from gene chip. The invention further provides application of the pattern recognition receptors RIG-I in preparation of anticancer drugs; the application specifically refers to combination between the pattern recognition receptors RIG-I and interferon alpha. The invention provides a new idea for oncotherapy.

Description

Application of pattern recognition receptor RIG-I in preparation of antitumor drugs

Technical Field

The invention relates to the field of biotechnology and medical diagnosis, in particular to application of a pattern recognition receptor RIG-I in preparation of antitumor drugs.

Background

During the infection of the body by pathogenic organisms, the innate immune response of the body is first activated. The activation of the innate immune response is mainly dependent on the recognition of specific components of pathogenic organisms by Pattern Recognition Receptors (PRRs) on the surface of immune cells of the body. PRRs found to date fall into three major categories: 1. TLRs (Toll-like receptors); 2. RLHs (RIG-I-like helicases); 3. NLRs (Nucleotide-oligomerization domain-like receptors). In the course of antiviral innate immune response, viral components such as DNA or RNA can be recognized by TLRs and RLHs of immune cells, thereby activating downstream signaling pathways, promoting immune cells to activate and express pro-inflammatory cytokines and type I interferons, increasing the antiviral activity of cells and promoting apoptosis of virus-infected cells, thereby playing a role in eliminating viral infection (Takeuchi, O et al, MDA5/RIG-I and virus recognition. curr Opin immunol.2008,20: 17-22).

RLHs are the major receptors for intracellular recognition of viral RNA, and there are two major members: RIG-I (retinoic acid-induced gene I) and MDA5(melanoma differentiation-associated gene 5). The molecular structure of RLHs consists of two parts: n-terminal CARDs (caspase-harvesting domains) and C-terminal RNA helicase domains. The RNA helicase domain is responsible for recognition of viral RNA, and its structure is similar to Dicer enzyme. The CARDs domain is primarily responsible for binding to downstream linker molecules, which in turn, transduce activation signals. Under the condition of virus infection or I-type interferon stimulation, the expression of RLHs can be obviously activated, thereby playing the role of enhancing the recognition of virus RNA and activating the downstream antiviral signal pathway of the virus RNA, and achieving the aim of eliminating virus infection (Mellan E, etc., Toll-like receptors and RNA resonators: two parallel walls to three anti viral responses. mol cell.2006,22: 561-.

Previous studies suggest that RIG-I primarily recognizes dsRNA, the product of viral replication. However, current studies indicate that viral RNA phosphorylated at the 5' end is the primary ligand recognized by RIG-I. In fact, 5 'end of most RNA derived from intracellular infectious virus is modified by phosphorylation, and RIG-I distinguishes self-derived and non-self-derived RNA by identifying whether the 5' end of the RNA has phosphorylation modification. However, the RNA expressed by the body itself originally has 5' phosphorylation modification, but the expression of the self RNA requires cleavage or modification in the nucleus, for example, mRNA requires methylation and capping modification at the 5' end, tRNA requires cleavage at the 5' end, and ribosomal RNA requires binding to ribosomal protein. Thus, under normal conditions, no body-derived RNA will activate RIG-I signaling in the cytoplasm. However, there are still a few 5' -phosphorylated self-RNAs in the organism, and the mechanism how these self-RNAs achieve escape from RIG-I recognition is not well understood (Yoneyama M et al, Function of RIG-I-like receptors in antiviral immunity. J Biol chem.2007,282: 15315-15318). In addition, RIG-I and MDA5 are the same as the receptors for RNA recognition by viruses of the RLHs family, but they recognize different types of viruses. The viruses mainly recognized by RIG-I include Vesicular Stomatitis Virus (VSV), Newcastle Disease Virus (NDV), Sendai virus (SeV), influenza virus (influenza virus), Japanese Encephalitis Virus (JEV), and the like; MDA5 recognizes primarily picornaviruses, including encephalomyocarditis virus (EMCV), encephalomyelitis virus (Theiler's virus), and Mengo virus (Mengo virus) (Kato H et al, Differential roles of MDA5and RIG-I viruses in the registration of RNA viruses. Nature.2006,441: 101-.

After RIG-I recognizes viral RNA through its C-terminal helicase domain, the molecular conformation is altered, exposing the N-terminal CARDs domain, and binding with the downstream linker molecule IPS-1 (IFN-. beta.promoter stimulator) located on the surface of mitochondrial membrane. IPS-1, also known as mavs (mitochondral anti-viral signaling) or VISA (virus-induced signaling adapter), binds to TRAF3 (tumor necrosis factor receptor-associated factor 3) upon activation and activates two major signaling pathways downstream: firstly, the expression of proinflammatory cytokines is activated by combining FADD (Fas-associated death domain-associating protein) to further activate a downstream transcription factor NF-kB; secondly, TBK-1 (TANK binding kinase-1) is activated to further activate downstream IRF3 and IRF7, so as to promote the cell to express type I interferon (Takeuchi et al, MDA5/RIG-I and virus recognition. curr Opin Immunol.2008,20: 17-22).

RIG-I signal pathways are precisely regulated by intracellular regulatory proteins and are mainly classified into positive regulatory proteins and negative regulatory proteins. The forward regulatory protein is generally directly involved in the activation of RIG-I signal, and plays a role in enhancing RIG-I signal and inhibiting virus replication, such as TRIM25 (tripartite motif 25), RNFl35 (ring-finger protein 135) and the like. The negative phase regulatory protein plays a role in inhibiting RIG-I signal conduction and avoids inflammatory injury caused by over-activation of RIG-I signals. It has been found that there are dozens of Negative regulatory proteins in the RIG-I signaling pathway, which are mainly divided into intracellular regulatory proteins derived from the expression of the body itself and protein molecules derived from the expression of viruses (Komuro A et al, Negative regulation of cytoplasmic RNA-mediated antiviral signaling. cytokinase. 2008,43: 350-358). The former mainly includes LGP2(laboratory of genetics and physiology-2), A20, Pinl, SIKE (supply of IKK epsilon), Atg5-Atg12, RNFl25(ring-finger protein125), DUBA (deubiquitting enzyme A), CYLD (cyclothymis), NLRxl (nuclear-binding domain and leucoine-rich X1), gClqR and ISG15(IFN stimulated gene15), etc. The latter is expressed by the virus itself and can evade and inhibit recognition and clearance of the host's antiviral innate immunity through a number of subtle mechanisms. At present, it has been found that part of viruses recognized by RLHs can negatively regulate the downstream signal path of the RLHs by using self-expressed proteins, and inhibit the generation of interferon, thereby realizing immune escape. The method specifically comprises the following steps: the non-structural protein NSl of influenza virus is capable of directly binding to RIG-I and inhibiting its function; the NS3-4A protein expressed by Hepatitis C Virus (HCV) and the 3ABC protein expressed by Hepatitis A Virus (HAV) can cleave IPS-l; the VP35 protein of Ebola virus (Ebola virus) and NS1 and NS2 proteins of respiratory syncytial virus can inhibit the phosphorylation of IRF 3; vaccinia virus (VACV) expressed E3L, N1L and K7R were able to inhibit RIG-I recognition and downstream signaling (Bowie AG et al, Viral evolution and version of pattern-recognition receptor signalling. Nat Rev immunization. 2008,8: 911-.

Activation of the RIG-I signal pathway is an important way for the expression of type I interferon of the body, plays a key role in anti-virus innate immune response, and is an important signal way for the body to resist virus infection. Currently, studies on the regulatory mechanisms of the RIG-I signaling pathway and the interaction of viruses with the host RIG-I signal are the focus and focus of innate immune responses and even immunological studies. The regulation and control mechanism of the RIG-I signal is clear, and has important significance for deeply understanding the anti-virus innate immune response of an organism, exploring the pathogenesis process of virus infectious diseases and the interaction relationship between the virus infectious diseases and the organism, and achieving the purpose of preventing and treating viral diseases.

Since cancer is one of the major diseases that endanger human health, there is now an increasing interest in the early diagnosis and prognosis of tumors in order to effectively treat and prevent tumors (e.g., hepatocellular carcinoma).

Taking primary liver cancer (i.e. primary Hepatocellular carcinoma (HCC)) as an example, the disease is a common malignant tumor in China, and the mortality of patients is third in all malignant tumors. According to the statistics of the world health organization, about 60 ten thousand HCC patients are newly added in the world every year, and the number of people dying from liver cancer every year is about 60 ten thousand. The development of HCC is a multistep, progressive process closely associated with chronic hepatitis and cirrhosis. About 1.2 hundred million hepatitis B virus carriers in China account for 1/3 worldwide infectious people. The recurrence rate of HCC radical operation in 5 years is about 60-70%, wherein the recurrence rate of 1 year after operation is up to 51.4-72.3%, and the recurrence rate of 5 years after the radical resection of small liver cancer is up to 30-40%. Therefore, postoperative recurrence and metastasis have become major obstacles to further improvement of the therapeutic effect of liver cancer.

Treatment methods such as postoperative perfusion chemotherapy metastasis and the like may reduce the postoperative recurrence rate of liver cancer patients, but some long-term studies show that the survival rate of patients after the postoperative chemotherapy is reduced relative to patients without the postoperative chemotherapy. These phenomena suggest that patients with a higher risk of postoperative recurrence and metastasis may benefit from these postoperative treatments, but patients with a lower risk of postoperative recurrence and metastasis may be counterproductive to these postoperative treatments. If the patient with high postoperative recurrence risk can be predicted by an effective prediction method, the recurrence rate can be reduced and the survival time of the patient can be prolonged by a series of intervention and prevention measures.

Therefore, how to effectively diagnose and intervene in the development of HCC and adaptively select a method for treating HCC patients is an urgent and important issue.

There has been a long history of studies using clinical indices (e.g., TNM grading, degree of cirrhosis) and single molecular indices (e.g., AFP, MMP) to predict recurrent metastasis of liver cancer. However, patients with similar clinical indicators or pathological types have distinct clinical outcomes, and therefore it is difficult to predict or evaluate patients with clinical indicators or individual molecular markers to achieve satisfactory results. The individual and predictive treatment of the tumor is helpful for further understanding clinical pathological characteristics and improving the clinical treatment of patients, thereby improving the tumor-free survival period and the absolute survival period of tumor patients. Determining liver cancer related genes and their involvement in liver cancer pathogenesis can provide a basis for individual predictive treatment of liver cancer and provide targets for new treatment schemes, thereby being beneficial to improving the cure rate of HCC (Thorgeirsson, S. et al, Hunting for tumor suppressor genes in liver cancer, Hepatology.2003;37: 739-.

Currently, molecular modeling, reviewed by tumor gene expression patterns, has been used as a new molecular standard for determining prognosis of different cancer types, including breast, prostate, lung, and brain tumors.

In 1999, Tamayo et al first studied acute leukemia by gene chip technology, typed leukemia according to the gene expression profile of tumor, and predicted the tumor type of new patients according to the established model, thus pioneering classification and prediction of tumors by gene chip (Tamayo, P. et al, interpretation patterns of gene expression with self-organizing maps: methods and applications of hematopoietic differentiation.) Proc Natl Acad Sci U S A.1999;96: 2907-12). Since then, similar methods were also used for analytical prediction of diffuse large B-cell lymphoma and breast cancer. These findings demonstrate that gene chip technology can more accurately classify tumors at the molecular level and predict tumor subtype, prognosis, and responsiveness to treatment.

Van't Veer et al, 70-gene-based breast cancer prognosis diagnosis models and related diagnosis chips Mammqaprint are approved by FDA to be on the market, and show good prospects for gene disease typing and prognosis judgment based on a gene expression spectrum model. However, recent studies of microarray-based prediction of early recurrence of HCC due to gene expression have only reported intrahepatic recurrence within 1 year in a small group of patients and used a gene chip of 6000 genes (Matoba, K. et al, Tumor HLA-DR expression linking to early intrahepatic involvement of hepatocellular cancer. Int J cancer.2005;115(2): 231-40). This increases the complexity of prognosis of HCC and decreases the operability.

Although it is known in the art that certain pattern recognition receptors have some correlation with tumors, such as Activation of TLR4signaling contributes to gastric cancer progression (X, Yuan et al, Activation of TLR4signaling proteins by induced mitochondral ROS production, cell Death and disease.2013;4, e794), helping lung cancer cells achieve immune escape (Weigan, H. et al, TLR4signaling proteins by immune system of human lung cells by induced immune regulation and apoptosis resistance. molecular immunology 2007;44 (11: 50 2859); TLR3 exerts a pro-apoptotic effect in breast cancer cells (Salaun B. et al, TLR3can directed trigger apoptosis in human cancer cells. J Immunol.2006;176: 4894-. However, the different functions of PRRs known in the art, and the difficulty in screening for specific PRRs that are tumor-associated and can be used as indicators of pathogenesis, treatment regimen selection, and prognosis, is relatively great.

At present, no research report about the relevance of a pattern recognition receptor RIG-I and tumors exists at home and abroad.

There is an urgent need in the art to find and use PRR molecules that can be effectively used for tumor diagnosis, tumor treatment protocol selection, and tumor prognosis evaluation.

Disclosure of Invention

The invention aims to find out a PRR molecule which can be effectively used for tumor diagnosis, tumor treatment scheme selection and tumor prognosis evaluation, and the invention also aims to provide the application of the PRR molecule in preparing anti-tumor medicaments.

The invention screens out the expression correlation of tumor (especially liver cancer) and the molecular-pattern recognition receptor RIG-I with the regulation and control function on the growth of cancer cells from the gene chip, thereby further providing the new application of the RIG-I molecule in the tumor diagnosis, the tumor treatment scheme selection and the tumor prognosis evaluation of the object.

The invention provides application of a pattern recognition receptor RIG-I in preparation of an anti-tumor medicament.

The pattern recognition receptor RIG-I is from: human, rat, mouse, dog, horse, cow, rabbit, or monkey, etc.

The pattern recognition receptor RIG-I, Gene ID: 23586.

The application of the pattern recognition receptor RIG-I in preparing the antitumor drugs specifically refers to the combination of the pattern recognition receptor RIG-I and interferon alpha (IFN-alpha).

The application of the pattern recognition receptor RIG-I in preparing the antitumor drugs specifically refers to the combination of a reagent for improving the expression level of the pattern recognition receptor RIG-I and interferon alpha (IFN-alpha).

The agent for improving the expression level of the pattern recognition receptor RIG-I comprises but is not limited to: RIG-I molecules, compositions containing RIG-I molecules as active substances, vectors (e.g., plasmids) containing RIG-I molecules, and the like.

The specific administration mode of the reagent for improving the expression level of the pattern recognition receptor RIG-I can adopt a direct naked DNA injection method, a liposome-encapsulated DNA direct injection method, a gold-encapsulated DNA Gene gun bombardment method, a plasmid DNA method carried by reproduction-defective bacteria, a DNA method carried by replication-defective adenovirus, a PEG modified protein drug injection method, a liposome-encapsulated protein intravenous injection method, a protein microsphere preparation subcutaneous injection method and the like (Hickman MA. et al, Gene expression direction information of DNA in vivo. hum Gene therapy apparatus.1994; 12: 1477-protein injection 1483; Siddhesh D. et al, DNA-based thermal and DNA delivery systems: A complex read therapy. AAPS journal.2005; 7: 61-77; Boulikas T. et al, nucleic acid amplification therapy for the protein injection 1.1997; Journal DNA of 1. 309. Gene injection method).

The RIG-I molecule comprises genes and proteins. The RIG-I gene is transcriptionally translated in a subject into a RIG-I protein product.

The tumor is specifically liver cancer, and the liver cancer is selected from: primary liver cancer, hepatocellular carcinoma, cholangiocellular carcinoma, metastatic liver cancer, secondary liver cancer and the like.

The results of cell experiments and animal experiments show that: in HCC cell lines SMMC-7721 and BEL-7402, the interference of RIG-I expression can inhibit IFN-alpha from proliferating cells and induce apoptosis. RIG-I can assist IFN-alpha to play a role in inhibiting cancer in vitro, thereby prompting that tumor patients with high RIG-I expression probably have better response to IFN-alpha treatment.

The injection of cholesterol-coupled RIG-I siRNA inside tumor significantly affected the inhibition of IFN-alpha on the growth of tumor and the content of AFP in serum. RIG-I assists IFN-alpha to play a role in inhibiting cancer in vivo, thereby prompting that tumor patients with high RIG-I expression probably have better response to IFN-alpha treatment.

The research shows that the invention is suitable for optimizing the curative effect of interferon alpha on treating tumor by adopting the treatment method of improving the amount of RIG-I molecules for tumor patients and patients with poor tumor prognosis.

The level of RIG-I molecules is at least 10-50%, preferably 20-40%, more preferably 30-35% higher than that of a normal control value.

The normal control values are: a level of RIG-I molecules measured in a non-tumor normal biological sample (e.g., a sample obtained from non-tumor cancer-adjacent tissue or normal tissue of the subject), a population standard level determined by statistics, or a normalized level.

In the present invention, the normal control value is a value calculated by qRT-PCR detection using T test in SPSS17.0, specifically 56.

The inventor of the invention has found through long-term and intensive research that: the expression of RIG-I molecules in tumor tissues of tumor patients is obviously reduced, the reduced expression has correlation with the survival time of the patients, and the low expression is obviously correlated with the poor prognosis of the patients, thereby confirming that the RIG-I molecules play an important role in tumor progression and patient prognosis. The inventor conducts further research on the basis of the above steps and finds that: RIG-I molecule can effectively inhibit the reproduction and growth of tumor cells in vivo and in vitro, thereby playing the role of anti-tumor. Thus, the present inventors have found a novel use of RIG-I molecules for tumor development judgment, treatment protocol selection and/or prognosis evaluation in a subject, and have completed the present invention on this basis.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

Detection reagent

As used herein, the terms "detection reagent" or "reagent for detecting a RIG-I molecule" or "reagent for detecting the amount of expression of a pattern recognition receptor RIG-I in a biological sample" are used interchangeably and refer to reagents that are specific for a RIG-I molecule and that can be used to directly or indirectly detect the presence and/or amount of a RIG-I molecule.

Since the sequence of RIG-I molecules is known in the art, one of ordinary skill in the art can prepare or obtain commercially agents specific for RIG-I molecules based on routine means. For example, detection reagents useful in the present invention include, but are not limited to: probes, gene chips, or PCR primers with detection specificity for RIG-I molecules, such as the antisense sequences of RIG-I molecules, the sequences shown in SEQ ID NOS: 2-4 used in the examples of the present invention, oligonucleotides, or other primer sequences.

To facilitate detection, the detection reagents of the invention may also carry detectable labels including, but not limited to: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens), and the like.

The detection reagents of the invention can be present in solution, immobilized on a support (e.g., substrate, adsorbate), or in other ways conventional in the art, so long as the presence is suitable for the detection of RIG-I molecules in a biological sample. For example, when the detection reagent of the present invention is a nucleotide probe, it may be present in the form of a biochip (or "microarray").

Detection kit and biological sample

The invention also provides a detection kit, which comprises: (i) detecting an effective amount of one or more reagents that detect a RIG-I molecule; (ii) one or more selected from the group consisting of: containers, instructions for use, positive controls, negative controls, buffers, adjuvants or solvents, such as solutions for suspending or immobilizing cells, detectable labels or labels, solutions for facilitating hybridization of nucleic acids, solutions for lysing cells, or solutions for nucleic acid purification.

The kit of the invention may also be accompanied by instructions for use of the kit, which describe how to use the kit for detection, and how to use the detection results for the determination of tumor development, selection of treatment regimens, and/or assessment of prognosis.

With the kit of the present invention, RIG-I molecules can be detected by various methods selected from the group consisting of, but not limited to: real-time quantitative reverse transcription PCR, biochip detection method, southern blotting, northern blotting or in situ hybridization. The detection mode can be adjusted and changed by those skilled in the art according to actual conditions and needs.

The biochip detection method, southern blotting, northern blotting and in situ hybridization can be described in "molecular blotting technique" Liu Kongqi et al, eds, published by chemical industry Press.

Of course, the kit may also contain other reagents that are clinically useful in the determination of tumor development, selection of treatment regimens, and/or prognostic assessment in a subject, to aid or validate the results obtained by detecting a RIG-I molecule. One of ordinary skill in the art can routinely select the desired compound according to particular needs.

As used herein, the terms "biological sample" or "test sample" are used interchangeably and refer to a sample obtained from a subject and used for the detection of RIG-I molecules. The biological sample may be fresh tissue, formalin-fixed or paraffin-embedded tissue, body fluid, blood, or cells, etc., preferably fresh tissue, formalin-fixed or paraffin-embedded tissue, obtained from a subject. These samples may be in the form of sections, smears, suspensions, solutions, RNA extracts, etc. suitable for detection, for example, total RNA may be extracted from tissues or cells prior to detection.

Tumor development judgment, antitumor drug preparation and/or prognosis evaluation

Without being limited by the following principles: the inventor finds that RIG-I molecules can effectively inhibit the propagation and growth of tumor cells in vivo and in vitro, thereby playing the role of resisting tumors. It is thus presumed that the decrease in the level of RIG-I molecules has a close relationship with the occurrence of tumors (and thus can be used to determine whether a tumor has already been developed), treatment (which can be used to specifically increase the level of RIG-I molecules in a patient), and prognosis, and thus can be used as a marker for the determination of tumor development, the preparation of antitumor drugs, and/or the evaluation of prognosis.

Generally, the detection kit of the present invention can be used for tumor development judgment, antitumor drug preparation and/or prognosis evaluation by the following methods: (a) obtaining a test sample from a subject; (b) contacting a sample to be detected with a detection reagent in the detection kit of the invention; (c) detecting the level of RIG-I molecule in the test sample and comparing the level to a control level; (d) and (3) carrying out tumor development judgment, antitumor drug preparation and/or prognosis evaluation according to the detection result: if the test result shows that the level of the RIG-I molecule in the tissue of the subject is lower than the control level, it indicates that the subject has a tumor, is suitable for treating the tumor by a treatment method for increasing the amount of the RIG-I molecule, or has poor tumor prognosis.

As used herein, the term "control level" refers to the level of RIG-I molecules used as a reference, including but not limited to: a level of RIG-I molecules measured in a non-tumor normal biological sample from the same subject (e.g., a sample obtained from non-tumor cancer-adjacent tissue or normal tissue of the subject), a population standard level determined by statistics, or a normalized level.

Useful for suggesting that the subject has a tumor, is suitable for treating a tumor with a treatment that increases the amount of a RIG-I molecule, or has a poor level of tumor prognosis may be: the level of RIG-I molecules in the test sample is 10-50%, preferably 20-40%, more preferably 30-35% lower than the control level.

Therapeutic methods to increase the amount of RIG-I molecules include, but are not limited to: administering to the patient an effective amount of a RIG-I molecule, or a composition comprising an active agent as described above. These substances can be administered to a subject by, for example and without limitation: direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-encapsulated DNA gene gun bombardment, plasmid DNA carried by reproduction-defective bacteria, or target DNA carried by replication-defective adenovirus.

As used herein, the term "prognosis" refers to the prediction of the likely course and outcome of a disease, which includes the judgment of the specific outcome of the disease (e.g., recovery, the appearance or disappearance of other abnormalities, such as certain symptoms, signs, and complications, and death). Poor prognosis as described in the present invention includes, but is not limited to: the survival period is shortened, the liver cirrhosis is easy to occur, the number of tumors is increased, the tumor enlargement is accelerated, the occurrence ratio of the portal vein cancer embolus is increased, the TNM grade is increased, and the like. After predicting the patient's prognosis, the patient's prognosis can be improved in combination with a treatment that increases the amount of RIG-I molecule.

The invention has the beneficial effects that:

the invention discloses the new application of RIG-I molecules in tumor development judgment, antitumor drug preparation and/or prognosis evaluation, and provides a new thought and approach for research, development and utilization of RIG-I-like receptors and even pattern recognition receptors;

the RIG-I molecule can be effectively used for tumor development judgment, treatment scheme selection and/or prognosis evaluation, so that a novel tumor diagnostic agent and/or therapeutic agent is provided for the field, and the RIG-I molecule has a certain clinical application prospect.

Drawings

FIG. 1: correlation of RIG-I expression in HCC with patient survival time, control patients in group 1, group 2, i.e., patients not receiving IFN- α treatment, were divided into two groups of high RIG-I expression and low RIG-I expression, with the median RIG-I expression being the median, where:

FIG. 1A is a Kaplan-Meier survival curve for tumor-free survival time;

FIG. 1B is a Kaplan-Meier survival curve for overall survival time;

the P value was calculated using the log-rank test in SPSS 17.0.

FIG. 2: correlation of RIG-I expression in HCC with the efficacy of IFN- α treatment in a patient, wherein:

FIG. 2A is a Kaplan-Meier survival curve for the overall survival time of group 1 patients, RIG-I high expressing patients, RIG-I low expressing patients;

FIG. 2B is a Kaplan-Meier survival curve for the overall survival time of group 2 patients, RIG-I high expression patients, RIG-I low expression patients;

the P value was calculated using the log-rank test in SPSS 17.0.

FIG. 3: effect of RIG-I expression on IFN- α treatment in vitro HCC experimental models. Transient transfection of control RNA and RIG-I small interfering RNA followed by IFN- α treatment in hepatoma cell lines SMMC-7721, BEL-7402, where:

FIG. 3A shows the results of MTT assay of cell proliferation;

FIGS. 3B and 3C are the results of apoptosis by flow cytometry;

results are shown as mean ± standard deviation (n = 4); p < 0.05; p < 0.01.

FIG. 4: effect of RIG-I expression on IFN- α treatment of HCC experimental models in vivo. Wherein,

FIG. 4A is a plot of tumor growth after intratumoral injection of cholesterol coupled control RNA and RIG-I interfering RNA, subcutaneous injection of PBS or IFN- α, in a tumor-bearing mouse model SMMC-LTNM;

FIG. 4B shows the AFP content in serum detected by ELISA;

FIG. 4C is the tumor growth curve of nude mice inoculated subcutaneously with SMMC-7721 normal cell line and RIG-I interference stable-sieve cell line, injected subcutaneously with PBS or IFN-alpha;

results show mean ± standard deviation (n = 4); p < 0.05; p < 0.01.

FIG. 5: RIG-I efficiently helped the generation of apoptosis-related IFN- α -inducible genes in vitro, control RNA and RIG-I small interfering RNA were transiently transfected in liver cancer cell lines SMMC-7721, BEL-7402, and expression of TRAIL, PML, XAF1, OAS1 was detected after IFN- α stimulation, showing mean ± standard deviation (n = 4); p < 0.05; p < 0.01.

FIG. 6: RIG-I efficiently aids in the production of apoptosis-related IFN-alpha inducible genes in vivo, wherein,

FIG. 6A shows the expression of TRAIL and PML after IFN- α stimulation by intratumoral cholesterol coupled control RNA and RIG-I interfering RNA injected into a tumor-bearing mouse model SMMC-LTNM;

FIG. 6B shows the expression of TRAIL and PML after IFN- α stimulation in normal primary liver cells of wild-type and RIG-I deficient mice;

FIG. 6C shows the expression of TRAIL and PML in liver cancer tissue after IFN-alpha stimulation in wild type and RIG-I deficient mice with DEN induced HCC;

results show mean ± standard deviation (n = 4); p < 0.05; p < 0.01.

Detailed Description

The present invention will now be described in detail with reference to examples and drawings, but the practice of the invention is not limited thereto.

The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), either according to conventional conditions or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1: preparation of detection kit

A detection kit suitable for detecting the expression of RIG-I in a biological sample by a real-time quantitative reverse transcription-PCR (qRT-PCR) method was prepared as follows:

(a) a vessel containing reverse transcriptase (200U/. mu.l);

(b) a container containing an RNase inhibitor (40U/. mu.l);

(c) containing reverse transcription 5 Xbuffer (75mM KCl,500mM Tris-Cl, pH8.3,25 ℃,3mM MgCl)₂10mM DTT);

(d) container with PCR upstream and downstream primers (10 μ M) for the RIG-I molecule of interest: RIG-I reverse transcription primers were: oligo (dT);

quantitative PCR primers:

5'-TGT GCT CCT ACA GGT TGT GGA-3' (upstream, SEQ ID NO:1) and

5'-CAC TGG GAT CTG ATT CGC AAA A-3' (downstream, SEQ ID NO: 2);

(e) vessel containing internal control reverse transcription primer and PCR upstream and downstream primers (10. mu.M): the reverse transcription reaction primer of the internal reference beta-actin is as follows: oligo (dT);

the quantitative PCR primers are as follows:

5'-ACA ATG AGC TGC TGG TGG CT-3' (upstream, SEQ ID NO: 3); and

5'-GAT GGG CAC AGT GTG GGT GA-3' (downstream, SEQ ID NO: 4);

(f) 10 XPCR buffer (50mM KCl,100mM Tris-Cl, pH9.0,25 ℃,1.0% Triton X-100);

(g) a vessel containing dNTPs (10 mM each);

(h) a vessel containing Taq DNA polymerase (3U/. mu.l);

(i) a container containing a fluorescent dye (SYBR I); and

(j) instructions for use.

Example 2: correlation of RIG-I expression in HCC with patient survival time

Two groups of 74 HCC control patients (not treated with IFN- α) collected in 1999 to 2006 were analyzed for expression of RIG-I protein levels in HCC tissues using immunohistochemistry. 74 HCC control patients were divided into high and low groups of 37 persons each, based on the median RIG-I expression. (group 1 tissue sections from hong Kong university, group 2 tissue sections from Shanghai Zhongshan Hospital)

The correlation of low expression of RIG-I with patient survival was analyzed separately and P values were calculated using log-rank test in SPSS17.0, and the results are shown in FIG. 1A and FIG. 1B, respectively.

As a result, it was found that: low expression of RIG-I was significantly associated with lower tumor-free survival time in patients (fig. 1A).

As a result, it was found that: low expression of RIG-I was significantly associated with lower overall survival time of patients (fig. 1B).

Cox regression analysis was performed on risk factors affecting HCC prognosis, and hazard ratios (95% confidence intervals) and P values were calculated using single and multifactorial Cox regression analysis in SPSS 17.0. Tables 1-2 show the results of single-factor and multi-factor Cox regression analyses affecting prognostic risk factors in HCC patients, where the HCC patients were 148 in total (same as fig. 1) in both groups. Age gender correction was used for multifactorial analysis.

TABLE 1 Single and multifactor Cox regression analysis of risk factors affecting tumor-free survival in control HCC patients

TABLE 2 Single and Multi-factor Cox regression analysis of the Risk factors affecting overall survival of control HCC patients

Low expression of RIG-I was found to be a significant independent risk factor predicting lower tumor-free survival and overall survival in HCC patients by single and multifactorial analysis.

The above results show that: the low expression of RIG-I in tumors of HCC patients is obviously related to the poorer prognosis of the patients, and the result indicates that the RIG-I is closely related to the occurrence and development of the HCC tumors, so that the RIG-I can be used as a marker for tumor development judgment, antitumor drug preparation and/or prognosis evaluation.

Example 3: correlation of RIG-I expression in HCC with the efficacy of IFN-alpha treatment in patients

Expression of RIG-I protein levels in tumor tissues of 151 HCC patients (including control and IFN- α treated patients) collected in 1999 to 2006 was analyzed using immunohistochemical methods. The 76 patients with HCC in group 1 and the 75 patients with HCC in group 2 were divided into two groups of high and low, with the median of RIG-I expression as a boundary.

The correlation of low expression of RIG-I with patient survival was analyzed separately and P values were calculated using log-rank test in SPSS17.0, and the results are shown in FIG. 2A and FIG. 2B, respectively. As a result, it was found that: low expression of RIG-I was significantly associated with lower overall survival of patients (figure 2A, B).

Cox regression analysis was performed on risk factors affecting HCC prognosis, and hazard ratios (95% confidence intervals) and P values were calculated using single and multifactorial Cox regression analysis in SPSS 17.0. Tables 3-4 show the results of single-factor and multi-factor Cox regression analyses affecting the prognostic risk factors in HCC patients, where the HCC patients were counted in 151 groups (same as in fig. 2). Age gender correction was used for multifactorial analysis.

TABLE 3 Cox regression analysis of the overall survival risk factors affecting RIG-I highly expressed HCC patients

TABLE 4 Cox regression analysis of RiG-I underexpressing HCC patients Overall survival Risk factors

Through single-factor and multi-factor analysis, the RIG-I high-expression HCC patients respond better to the postoperative IFN-alpha treatment, and only in the RIG-I high-expression patients, the postoperative IFN-alpha treatment is a significant independent risk factor for predicting the overall survival time of the HCC patients.

The above results show that: the high expression of RIG-I in the tumor of an HCC patient is obviously related to the better IFN-alpha reactivity of the patient, and the result indicates that the RIG-I is closely related to the IFN-alpha treatment after the HCC tumor operation, so that the RIG-I can be used as a marker for preparing an anti-tumor medicament and/or evaluating prognosis.

Example 4: RIG-I in vitro help IFN-alpha inhibit HCC cell growth

The following sequences (synthesized by Shanghai Jima, Inc.) were used to interfere with RIG-I expression in human HCC cell lines SMMC-7721, BEL-7402, respectively:

RIG-I siRNA：

5'-GGU GGA GGA UAU UGC AAC U

-3' (sense strand, SEQ ID NO:5)

5'-AGU UGC AAU AUC CUC CAC C3' (antisense strand, SEQ ID NO:6) control siRNA:

5'-UUC UCC GAA CGU GUC ACG U

-3' (sense strand, SEQ ID NO:7)

5'-ACG UGA CAC GUU CGG AGA A

-3' (antisense strand, SEQ ID NO:8)

The possibility of TT-overhangs at the 3' end of the above-mentioned SEQ ID NO 5-8 is a commonly used modification method in the synthesis of small RNA sequences, which has NO effect on their function and mainly plays a role in stabilizing nucleotides (Wilda, M. et al, Kiling of free cells with a BCR/ABL fusion gene by RNA interference (RNAi); oncogene.2002;21: 5176-5124).

Human HCC cell lines SMMC-7721 and BEL-7402 were purchased from Shanghai Biochemical cell institute. Small RNAs were transfected using INTERFERIN transfection reagent (Polyplus) at a final siRNA transfection concentration of 10nM, and the procedure was as described in standard procedures.

Human recombinant IFN-alpha-2 b was purchased from Keyin Probiotics at a stimulation concentration of 1000U/ml.

And (3) detecting cell proliferation: the proliferation of SMMC-7721 and BEL-7402 cells in vitro was detected by MTT assay. Will be 5X 10³SMMC-7721 and BEL-7402 cells were plated in each well of 96-well plate and cultured overnight, transfected with control RNA or RIG-I siRNA the next day, and assayed at 0, 48, and 96 hours after transfection for IFN-alpha stimulation 48 hours. First, the culture supernatant was discarded, and 100. mu.l of fresh medium containing MTT0.5mg/ml (Sigma Co.); then incubated at 37 ℃ for 4 hours; finally, 100. mu.l DMSO (Sigma) was added and shaken for 10 minutes. The final absorbance was measured using a wavelength of 570 nm.

And (3) detecting cell apoptosis: after 48 hours of transfection of the SMMC-7721 and BEL-7402 cells, IFN-alpha was stimulated for 0 to 72 hours, and the cells were harvested, washed, resuspended, and tested using Annexin V-FITC apoptosis test kit (Calbiochem, Inc.). Labeled cells were detected using a FACSCalibur flow cytometer and the data analyzed using CellQuest software (purchased from Becton Dickinson), annexin v (annexin v) positive and PI negative cells were considered apoptotic.

The results of the effect on the proliferation of SMMC-7721 and BEL-7402HCC cell lines after transfection of RIG-I siRNA are shown in FIG. 3A, and the results of the effect on the apoptosis of SMMC-7721 and BEL-7402HCC cells are shown in FIG. 3B.

The results show that: in HCC cell lines SMMC-7721, BEL-7402, interference with RIG-I inhibited IFN- α cell proliferation and induced apoptosis (FIG. 2).

The above results show that: RIG-I assists IFN-alpha to play a role in inhibiting cancer in vitro, thereby prompting that tumor patients with low RIG-I expression may have better response to IFN-alpha treatment.

Example 5: RIG-I assists IFN-alpha to inhibit growth of HCC cells in vivo

The in vivo tumor inhibition effect of RIG-I is observed by utilizing a human primary HCC tissue subcutaneous tumor-bearing nude mouse model SMMC-LTNM and liver cancer cell lines SMMC-7721 and RIG-I to interfere with the SMMC-7721 subcutaneous tumor-bearing mouse model.

Methods of model construction are described in the literature (Hou, J. et al, Identification of microorganisms in human liver and hepatocellular receptors miR-199a/b-3p as the therapeutic target for hepatocellular receptors cancer. cancer cell.2011,19:232-

Both cholesterol-coupled RIG-I siRNA and cholesterol-coupled control siRNA (i.e., RNA shown in SEQ ID NOS: 5-8) were synthesized by Shanghai Gilmar corporation (siRNA that can enter cells through cell membranes autonomously after cholesterol coupling and transfection efficiency is high (Wolfrem, C. et al, mechanistic and optimization of lipophilic siRNA delivery in vivo) Nat Biotechnol.2007; 25: 1149-1157.) when RNA is transfected in vivo, 10nmol (0.1ml) of cholesterol-coupled RIG-I siRNA or cholesterol-coupled control siRNA was directly injected intratumorally, once every three days, for two weeks.

The calculation of tumor volumes was performed as described in the literature (Qi, R, et al, Notch1signaling inhibition growth of human hepatoma through inhibition of cell cycle arrest and apoptosis Notch1 signaling. Cancer Res.2003; 63: 8323. sup.8329). Determination of serum AFP an AFP ELISA kit (purchased from Autobio) was used.

Human recombinant IFN-alpha-2 b was purchased from Keyin Probiotics, and the therapeutic concentration of subcutaneous injection in mice was 5X 10⁶U/Kg。

The results of experiments on the effect of RIG-I in vivo-assisted IFN-alpha on tumor cell growth and serum AFP content are shown in FIG. 4. The results show that: intratumoral injection of cholesterol-coupled RIG-I siRNA significantly affected the inhibitory effect of IFN- α on tumor growth and serum AFP levels (figure 4).

The results show that: RIG-I assists IFN-alpha to play a role in inhibiting cancer in vivo, thereby prompting that tumor patients with high RIG-I expression probably have better response to IFN-alpha treatment.

Example 6: RIG-I in vitro and in vivo assistance for IFN-alpha induction gene generation

Total RNA from cells and tissues was extracted by TRIzol (Invitrogen). qRT-PCR was performed on a LightCycler1.5 (Roche) real-time quantitative PCR instrument using the test kit of example 1. Relative quantification of IFN-alpha inducible genes 2^-ΔΔCtMethod (beta-actin is an internal reference) (Livak, KJ. et al, Analysis of relative gene expression data-time quantitative PCR and the 2-delta. Ct method, methods, 2001; 25: 402-.

RIG-I siRNA was transfected into human hepatoma cell lines SMMC-7721 and BEL-7402 (same as example 2), and expression of IFN-alpha activated downstream apoptosis-related genes TRAIL, PML, XAF1 and OAS1 was detected by qRT-PCR (FIG. 5).

Intratumoral injection of cholesterol-coupled control RNA and RIG-I interfering RNA (same as example 5) in a tumor-bearing mouse model SMMC-LTNM, expression of TRAIL, PML following IFN- α stimulation (FIG. 6A); expression of TRAIL, PML following IFN- α stimulation in normal primary liver cells from wild-type and RIG-I deficient mice (FIG. 6B); DEN induced HCC wild type and RIG-I deficient mice, IFN-alpha stimulation of liver cancer tissue in TRAIL, PML expression (figure 6C).

The results show that: RIG-I assists IFN-alpha downstream induction gene production in vitro and in vivo, thereby suggesting that tumor patients with high RIG-I expression may have better response to IFN-alpha treatment.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Claims (7)

1. Application of a pattern recognition receptor RIG-I in preparing antitumor drugs.

2. The use of the pattern recognition receptor RIG-I as claimed in claim 1, in the preparation of an anti-tumor medicament, wherein the use is the use of the pattern recognition receptor RIG-I in combination with interferon alpha.

3. The use of the pattern recognition receptor RIG-I as claimed in claim 1, wherein the use is the combination of an agent for increasing the expression level of the pattern recognition receptor RIG-I and interferon- α.

4. The use of the pattern recognition receptor RIG-I as claimed in claim 3, wherein the agent for increasing the expression level of the pattern recognition receptor RIG-I comprises RIG-I molecule, a composition containing RIG-I molecule as active substance, and a carrier containing RIG-I molecule.

5. The use of the pattern recognition receptor RIG-I as claimed in claim 4, wherein the RIG-I molecule comprises RIG-I gene and RIG-I protein.

6. The use of the pattern recognition receptor RIG-I as claimed in any one of claims 1 to 5 in the preparation of an anti-tumor medicament, wherein the tumor is liver cancer.

7. The application of the pattern recognition receptor RIG-I in preparing an antitumor medicament as claimed in claim 6, wherein the liver cancer is primary liver cancer, hepatocellular carcinoma, cholangiocellular carcinoma, metastatic liver cancer or secondary liver cancer.

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