CN118086495A - Application of SP1 gene as biomarker and therapeutic target of drug-resistant solid tumor - Google Patents
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Abstract
The invention discloses application of an SP1 gene as a biomarker and a therapeutic target point of drug-resistant solid tumors, and particularly discloses novel discovery of drug resistance of solid tumors based on high expression of SP1 in solid tumor tissues, wherein the gene or/and protein is used as the target point for diagnosing and treating the drug-resistant solid tumors.
Description
Technical Field
The invention relates to a solid tumor drug resistance diagnosis and prognosis evaluation technology, in particular to a novel discovery that a Sp1 transcription factor (SP 1, namely Sp1 transcription factor) is highly expressed in a solid tumor tissue and causes solid tumor drug resistance, and the gene or/and protein is used as a target point for diagnosing and prognosing the solid tumor drug resistance.
Background
Conventional treatments for solid tumors include surgery, chemotherapy, and radiation therapy. Chemotherapy (chemotherapy) plays an important role in the treatment of solid tumors, however, most patients experience chemotherapy drug tolerance during the course of receiving chemotherapy resulting in treatment failure. More importantly, in chemotherapy, the tumor develops resistance to one drug (single drug resistance) and also develops cross resistance to other drugs which are not used and have different chemical structures and action mechanisms, and this phenomenon is called multi-drug resistance. Research on the mechanism of drug resistance occurrence of solid tumors and the search for and development of drugs for reversing drug resistance are important research fields in current tumor prevention and treatment. In order to reverse the drug resistance of solid tumors, improve the curative effect of chemotherapy of solid tumors, reduce the drug resistance risk and prolong the survival rate, the field is urgently required to research the mechanism and key gene targets of the drug resistance occurrence of solid tumors in various modes.
Disclosure of Invention
Based on the research findings of the invention, in one aspect, the invention provides the application of the SP1 gene or the coded protein thereof as a target in preparing a reagent or a kit for diagnosing and/or evaluating the drug resistance of solid tumors; the solid tumor is gastric cancer or intestinal cancer.
The invention also provides application of the SP1 gene or the detection reagent of the coded protein thereof in preparing a reagent or a kit for diagnosing and/or prognosis evaluating solid tumor drug resistance; the solid tumor is gastric cancer, intestinal cancer, esophageal cancer, brain low-grade glioma, liver cancer and pancreatic cancer.
Alternatively, the reagent or kit detects transcribed messenger RNA of the SP1 gene or the SP1 gene-encoded protein by RT-PCR, real-time quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or biological mass spectrometry, in situ hybridization, northern blotting, chip, high throughput sequencing platform, immunohistochemistry or enzyme-linked immunosorbent assay. Further alternatively, the kit or kit contains a specific primer for amplifying the SP1 gene, a probe that hybridizes to the nucleotide sequence of the SP1 gene, or an antibody or antibody fragment that specifically binds to the SP1 protein. The antibody is a monoclonal antibody or a polyclonal antibody.
Alternatively, the test sample of the reagent or the kit is serum, plasma, cells, cell culture supernatant, urine, tissue or tissue lysate.
The invention also provides application of the SP1 gene or the coded protein thereof serving as a target point in preparing a drug for treating drug-resistant solid tumors; the solid tumor is gastric cancer or intestinal cancer.
The invention further provides application of the SP1 gene expression inhibitor or the SP1 gene encoding protein expression inhibitor in preparing medicines for treating drug-resistant solid tumors; the solid tumor is gastric cancer, intestinal cancer, esophageal cancer, brain low-grade glioma, liver cancer and pancreatic cancer.
Alternatively, the SP1 gene expression inhibitor is selected from a CRISPR gene editing therapeutic agent, an antisense nucleic acid agent, an siRNA agent or a miRNA agent that blocks the normal transcription or post-transcriptional translation process of the SP1 gene by CRISPR/Cas9 gene editing techniques or RNA interference techniques.
Alternatively, the inhibitor of SP1 protein expression is selected from the group consisting of protein glycosylation inhibitors, protein phosphorylation inhibitors, or neutralizing antibodies that affect post-translational modification of SP1 protein or affect SP1 protein stability, as well as affect SP1 protein expression levels, activity, or function.
Drawings
FIG. 1 shows the analysis of SP1 protein expression in gastric cancer chemotherapeutic resistant tissues by IHC staining in example 1; FIG. 1A is an immunohistochemical (Immunohistochemistry, IHC) staining micrograph of SP1 protein in Post-chemotherapy gastric cancer tissue (Post) and its paired Pre-chemotherapy gastric cancer tissue (Pre) of a representative 4-case gastric cancer patient undergoing Post-chemotherapy disease progression in example 1; FIG. 1B is a statistical plot of IHC scores of SP1 expression in post-chemotherapy gastric cancer tissue and paired pre-chemotherapy gastric cancer tissue of 31 gastric cancer patients receiving post-chemotherapy disease progression.
FIG. 2 is a micrograph of IHC staining of SP1 protein in cancer tissue after treatment with a chemotherapeutic agent after subcutaneous tumor formation of mouse gastric or intestinal cancer cells in example 2: FIG. 2A is a micrograph of IHC staining and a quantitative statistical chart of SP1 protein expression in subcutaneous tumor cancer tissues of two groups of mice after mice are subjected to subcutaneous tumor formation by using mouse gastric cancer cell MFC and then are subjected to intraperitoneal injection of normal saline or fluorouracil; fig. 2B is a micrograph of IHC staining and a quantitative statistical plot of SP1 protein expression in two groups of mouse subcutaneous tumor cancer tissues after mouse intestinal cancer cell CT26 has been subcutaneously tumor-developed and injected intraperitoneally with physiological saline or fluorouracil.
FIG. 3 is a graph showing the results of the expression levels of SP1 proteins in human gastric cancer-resistant cell lines SGC7901 ADR and SGC7901 VCR and in the control cell line SGC7901 in example 3.
FIG. 4 shows the expression changes of SP1 mRNA in human and mouse gastric or intestinal cancer cells sensitized to chemotherapeutic agents in example 4 under conditions induced by sub-lethal doses of chemotherapeutic agents: FIG. 4A shows comparison of SP1 mRNA expression levels of human gastric cancer cells SGC7901 on days 1, 3,5 and 7 under fluorouracil (1. Mu.g/mL) culture conditions with SP1 mRNA expression levels of untreated control cells, FIG. 4B shows comparison of SP1 mRNA expression levels of mouse gastric cancer cells MFC on days 1, 3,5 and 7 under fluorouracil (1. Mu.g/mL) culture conditions with SP1 mRNA expression levels of untreated control cells, and FIG. 4C shows comparison of SP1 mRNA expression levels of mouse gastric cancer cells CT26 on days 1, 3,5 and 7 under fluorouracil (1. Mu.g/mL) culture conditions with SP1 mRNA expression levels of untreated control cells.
FIG. 5 is a graph showing the efficiency of the RNA interference technique for knocking down SP1 gene in human gastric cancer cells in example 5: FIG. 5A is a statistical plot of SP1 mRNA expression from two cell lines (siSP #1 and siSP # 2) of human gastric cancer cells SGC7901 VCR and MKN45 with SP1 gene knockdown verified by real-time quantitative PCR; FIG. 5B is a schematic diagram showing the SP1 protein expression of two cell lines (siSP #1 and siSP # 2) obtained by Western Blotting (WB) after SP1 gene knockdown in human gastric cancer cells SGC7901 VCR and MKN 45.
FIG. 6 shows apoptosis rate of SP1 gene knockdown and control human gastric cancer cells of example 6 under chemotherapeutic treatment conditions: FIG. 6A is a statistical graph of apoptosis rate of SP1 gene knockdown and control human gastric cancer cell SGC7901 VCR under vincristine treatment conditions; fig. 6B is a statistical plot of apoptosis rate of SP1 gene knockdown and control human gastric cancer cell MKN45 under fluorouracil treatment conditions.
FIG. 7 is a correlation analysis of SP1 gene expression with survival of cancer patients by TCGA database analysis in example 7: FIG. 7A is a graph showing analysis of total survival of patients with low and high expression of SP1 gene in esophageal cancer, low-grade glioma in brain, liver cancer and pancreatic cancer; FIG. 7B is a graph showing the progression-free survival analysis of patients with esophageal cancer, low-grade glioma, liver cancer and pancreatic cancer, in which the SP1 gene is expressed under and under.
Detailed Description
Unless otherwise indicated, the terms or methods herein are understood or implemented using existing methods based on knowledge of one of ordinary skill in the relevant art.
The protein encoded by the SP1 gene is a zinc finger transcription factor that binds to GC-rich motifs of many promoters. The protein encoded by the SP1 gene is involved in many cellular processes including cell differentiation, cell growth, apoptosis, immune response, response to DNA damage and chromatin remodeling. The SP1 gene resides on human chromosome 12 and has a sequence region of about 36kbp with 3 transcriptional variants each encoding 3 different protein subtypes.
In the present application, the term "solid tumor resistance" refers to the resistance of a solid tumor patient or isolated solid tumor cell or tissue in vitro to a single chemotherapeutic agent or to multiple chemotherapeutic agents, and specifically refers to an increase in the IC50 (half inhibitory concentration, which refers to the concentration of drug required to reduce the number of surviving cells by a half after administration) of a chemotherapeutic agent by a factor of more than 2 after induction or other treatment, as compared to a parent cell that is sensitive to the chemotherapeutic agent (chemosensitivity refers to the inhibition of cell growth by a chemotherapeutic agent at a normal blood concentration of greater than 60%).
The invention relates to application of a detection reagent of an SP1 gene or a coded protein thereof in preparation of a reagent or a kit for prognosis evaluation of solid tumors, wherein the increase of the expression level of the SP1 gene or the coded protein thereof is an indication of poor prognosis of the solid tumors.
The term "detection reagent for SP1 gene or protein encoded thereby" is not to be construed as merely being a detection reagent for SP1 gene or protein encoded thereby in the present invention, but is intended to include detection reagents known to those skilled in the art which reflect the expression level of the detection reagent for SP1 gene or protein encoded thereby. For example, the amount of mRNA expressed can be indirectly detected by quantitatively detecting cDNA obtained by reverse transcription of the SP1 gene.
The detection reagent may be any reagent known to those skilled in the art, for example, a nucleic acid which hybridizes to the RNA and is labeled with a fluorescent label; the detection agent of RNA can be selected from primers of RT-PCR and primers for amplifying cDNA, which is the product of RT-PCR; in some embodiments, the detection reagent comprises a reagent suitable for use in at least one of the following methods: real-time fluorescent quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or biological mass spectrometry.
The term "antibody" includes polyclonal antibodies as well as monoclonal antibodies, and the term "antibody fragment" includes antigen compound binding fragments of such antibodies, including Fab, F (ab') 2, fd, fv, scFv, bispecific antibodies, and antibody minimal recognition units, as well as single chain derivatives of such antibodies and fragments, such as scFv-Fc, and the like. The type of antibody may be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional), humanized (humanized) antibodies, and human antibodies, as well as related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
In some embodiments, the measurement sample comprises blood (whole blood), serum, plasma, cell culture supernatant, urine, tissue, or tissue lysate.
The SP1 gene expression inhibitor refers to an agent, a preparation or a medicament for blocking the normal transcription or posttranscriptional translation process of an SP1 gene based on a CRISPR/Cas9 gene editing technology or an RNA interference technology, such as CRISPR gene editing therapeutic medicaments, antisense nucleic acid medicaments, siRNA medicaments, miRNA medicaments and the like.
The SP1 protein expression inhibitor refers to an agent, a preparation or a medicament, such as an protein glycosylation inhibitor, a protein phosphorylation inhibitor, a neutralizing antibody and the like, which influences the post-translational modification process of the SP1 protein or the stability of the SP1 protein and the expression level, activity or function of the SP1 protein.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: IHC staining analysis of SP1 protein expression in gastric cancer chemotherapy drug resistant tissue
1. Study object
Clinical samples of patients with gastric cancer chemotherapy resistance are taken from the Beijing digestive disease hospital, and the study is passed through the examination of the ethics committee of the Beijing hospital at the air force medical university, and the patients and family members sign informed consent.
2. Research method
The expression and spatial distribution of the SP1 protein on chemotherapy-resistant GC tissues are detected by adopting an IHC method, and the method comprises the following specific steps:
Placing the pathological paraffin sections into an oven at 60-65 ℃ for 2 hours, and then dewaxing; placing the sections in 10% formalin to fix the sections for 10min, and increasing the adhesion of the tissue sections; placing the fixed slice in antigen retrieval liquid for retrieval for 15min, cooling to room temperature, and cleaning a slide by using ddH 2 O; placing the trimmed slice in 3% H 2O2 water solution for 15min, and removing endogenous peroxidase; dripping blocking buffer (Akoya Biosciences) in the tissue sample area, ensuring that the tissue area can be completely covered, and placing in a moisturizing box for incubation for 10min; dripping diluted primary antibody working solution (rabbit anti-human SP1 monoclonal antibody, proteintech, 1:200), immersing a tissue area, and placing the tissue area into a moisturizing box at room temperature for incubation for 1h; removing residual liquid after TBST cleaning, dripping an opal polymer horseradish peroxidase (HRP) coupled donkey anti-rabbit IgG (Akoya Biosciences) into a slide, and incubating for 10min in a moisturizing mode; dropwise adding a chromogenic working solution (Opal 520,Akoya Biosciences) on the glass slide to submerge the tissue region for incubation for 10min; and (3) performing microwave treatment, removing the primary antibody and the secondary antibody, cleaning a slide by using a TBST solution, washing by using ddH 2 O, discarding washing liquid, dripping DAPI (Akoya Biosciences, 1:5) working solution on the slide, performing moisturizing incubation for 5min, dripping a fluorescent anti-quenching sealing tablet for sealing, fixing a cover slip by using a nail polish, acquiring a slide tissue sample area image by using a Vectra multispectral imaging system, and performing quantitative statistical analysis on the image by using inForm software.
3. Conclusion of the study
FIG. 1A is an immunohistochemical staining micrograph of SP1 protein in Post-chemotherapy gastric cancer tissue (Post) and its paired Pre-chemotherapy gastric cancer tissue (Pre) of a representative 4-case gastric cancer patient receiving Post-chemotherapy disease progression (Pt#1-4); FIG. 1B is a statistical plot of IHC scores (H-score) of SP1 expression in post-chemotherapy gastric cancer tissue and paired pre-chemotherapy gastric cancer tissue of 31 gastric cancer patients receiving post-chemotherapy disease progression. The results show that the SP1 protein in tumor cells of a chemotherapy-resistant patient has higher expression level than before treatment.
Example 2: IHC staining micrograph of SP1 protein in cancer tissue treated with chemotherapeutic drug after mouse gastric or intestinal cancer cells are subcutaneously tumorigenized
1. Study object
Female 615 mice and BALB/c mice were purchased from Shanghai laboratory animal research centers for 4-6 weeks; both MFC and CT26 cells were derived from the American Type Culture Collection (ATCC) and deposited by the national emphasis laboratory for the integrated control of digestive system tumors at the university of air force army medicine.
2. Research method
5-10X 10 5 MFC and CT26 cells were inoculated to the posterior side of 615 mice and BALB/c mice, respectively, to establish subcutaneous tumor models; after inoculation of tumor cells, mice were randomly assigned to different groups for treatment; the chemotherapeutic drug fluorouracil (MCE, 20 mg/kg) was administered intraperitoneally on day 3 post tumor inoculation, once every 3 days; mice are sacrificed after reaching the tumor growth end point, and the distribution of SP1 protein in cancer tissues after fluorouracil treatment in different mouse tumor tissues is detected by adopting an IHC method, wherein the specific staining step is the same as that of example 1.
3. Conclusion of the study
FIG. 2 is a IHC staining micrograph of SP1 protein in cancer tissue after treatment with a chemotherapeutic agent after subcutaneous tumor formation of gastric or intestinal cancer cells in mice. FIG. 2A is a micrograph of IHC staining and a quantitative statistical chart of SP1 protein expression in subcutaneous tumor cancer tissues of two groups of mice after mice are subjected to subcutaneous tumor formation by using mouse gastric cancer cell MFC and then are subjected to intraperitoneal injection of normal saline or fluorouracil (Fluorouracil); fig. 2B is a microscopic and quantitative statistical IHC staining of SP1 protein expression in cancer tissue of two groups of mice following subcutaneous tumor formation in mice, following intraperitoneal injection with normal saline or fluorouracil, with differences of significance (P < 0.01). The above results indicate that fluorouracil treatment induces increased SP1 protein expression in MFC and CT26 subcutaneous tumor cancer tissues.
Example 3: WB detection of expression difference of SP1 protein in gastric cancer cell SGC7901 and drug-resistant cell SGC7901 ADR、SGC7901VCR
1. Study object
The gastric cancer cell strain SGC7901 cells are purchased from Shanghai research biochemical reagent limited company, and drug resistant cells SGC7901 ADR and SGC7901 VCR are selected, constructed and maintained by exposing the SGC7901 cells to chemotherapeutics in the national key laboratory for digestive system tumor integration control of the air force medical university, and are preserved by the national key laboratory for digestive system tumor integration control of the air force medical university.
2. Research method
The expression level of the SP1 protein in SGC7901 and SGC7901 ADR、SGC7901VCR cells is detected by adopting WB, and the specific steps are as follows:
Extracting proteins from cells with RIPA buffer containing protease inhibitor (Roche) when the 3 cell densities increase to 80%; SDS-PAGE gel electrophoresis is adopted, and then the mixture is transferred to a nitrocellulose membrane; after hybridization with a second enzyme-labeled antibody (donkey anti-rabbit, sheep anti-mouse IgG, GE Healthcare Life Sciences, 1:5000) using primary antibodies to β -actin and SP1 (rabbit anti-human SP1 mab, CELL SIGNALING Technology,1:1000; mouse anti-human β -actin mab, santa Cruz Biotechnology, 1:2000) according to manufacturer recommended dilutions, the protein bands were visualized using a Bio-Rad ChemiDoc xrs+ imaging system as described by chemiluminescent detection kit (Pierce).
3. Conclusion of the study
FIG. 3 is a graph showing the results of the expression levels of SP1 proteins in human gastric cancer-resistant cell lines SGC7901 ADR and SGC7901 VCR and in a control cell line SGC 7901. The results indicated that the multi-drug resistant cell lines SGC7901 ADR and SGC7901 VCR had higher SP1 gene expression levels than the chemotherapeutic sensitive SGC7901 cell line.
Example 4: expression change of SP1 mRNA of gastric cancer or intestinal cancer cells under the induction condition of sub-lethal dose of chemotherapeutic medicine 1. Study object
Culturing gastric cancer cell SGC7901 as in example 3; MFC and CT26 cells were drawn from American Type Culture Collection (ATCC) and deposited by the national emphasis laboratories for digestive system tumor control at the university of air force medical science.
2. Research method
The expression changes of the SP1 gene were observed by simulating the in vivo chemotherapeutic environment of the tumor by administering a sub-lethal dose (drug concentration at which cells retain 50% of mortality after 24h of drug treatment) of the chemotherapeutic drug. The expression levels of the SP1 gene were detected by real-time quantitative PCR on days 1,3,5, 7 after treatment with sublethal doses of fluorouracil (MCE, 1. Mu.g/mL) on SGC7901, MFC and CT26 cells, as follows:
Extracting total RNA when the density of 3 cells is increased to 80%; then reverse transcribed into cDNA using PRIMESCRIPT RT kit (Takara); PCR was performed on a real-time fluorescent quantitative PCR detection system (Bio-Rad) using SYBR Premix Ex-Taq kit (Takara). The primer sequences for each PCR reaction are shown in Table 1.
TABLE 1 real-time quantitative PCR primer sequences
3. Conclusion of the study
FIG. 4 shows the variation of SP1 mRNA expression of gastric or intestinal cancer cells under sub-lethal dose of chemotherapy drug induction: FIG. 4A is a comparison of SP1 mRNA expression levels of human gastric cancer cell SGC7901 under fluorouracil (1. Mu.g/mL) culture conditions at days 1,3, 5, and 7 with SP1 mRNA expression levels of untreated control cells; FIG. 4B is a comparison of the expression level of SP1 mRNA in mouse gastric cancer cells MFC under fluorouracil (1. Mu.g/mL) culture conditions at days 1,3, 5, and 7 with the expression level of SP1 mRNA in untreated control cells; FIG. 4C is a comparison of SP1 mRNA expression levels of mouse gastric cancer cell CT26 on days 1,3, 5, and 7 under fluorouracil (1. Mu.g/mL) culture conditions with SP1 mRNA expression levels of untreated control cells. The results show that the chemotherapeutic drugs can induce the expression of the SP1 gene, and the longer the survival time (namely the stronger the drug resistance) under the chemotherapeutic condition, the more obvious the increase in the cells.
Example 5: efficiency verification after SP1 gene in human gastric cancer cells is knocked down by RNA interference technology
1. Study object
The drug-resistant gastric cancer cell SGC7901 VCR is the same as in example 3, the gastric cancer cell strain MKN45 is introduced from a BRCC Chinese microbial strain cell library and is preserved by the national key laboratory for digestive system tumor integration prevention and control of air force medical university.
2. Research method
Drug-resistant gastric cancer cells SGC7901 VCR and MNK45 are taken as target cells, siRNA is designed and synthesized, SP1 gene expression of the cells is knocked down, and real-time quantitative PCR and WB are adopted for verification. The specific experimental steps are as follows:
siRNA and oligonucleotides were transfected into SGC7901 VCR and MNK45 cells using JetPRIME reagents (Polyplus Transfection) according to the manufacturer's instructions. Designated cells were transfected and knock-out efficiency was verified by real-time quantitative PCR. Extracting total RNA after 24 hours of cell transfection, and carrying out real-time quantitative PCR in the same specific steps as in example 4; the WB procedure was as in example 3. The primer sequences of each PCR reaction are shown in Table 1, and the target sequences of the siRNA are shown in Table 2.
TABLE 2 siRNA sequences targeting the SP1 Gene
3. Conclusion of the study
FIG. 5 is a graph showing the efficiency of the RNA interference technique for knocking down SP1 gene in human gastric cancer cells. FIG. 5A is a statistical plot of SP1 mRNA expression of two cell lines (siSP #1 and siSP # 2) from which SP1 gene knockdown in human gastric cancer cells SGC7901 VCR and MKN45 were verified by real-time quantitative PCR, the differences were significant (P < 0.01); FIG. 5B is a schematic diagram showing the expression of SP1 protein in two cell lines (siSP 1#1 and siSP 1#2) of WB-verified human gastric cancer cells SGC7901 VCR and MKN45 after SP1 gene knockdown. The above results indicate that siRNA successfully interfered with SP1 gene expression, resulting in deletion of SP1 mRNA and protein expression.
Example 6: apoptosis rate of SP1 gene knockdown and control human gastric cancer cells under treatment condition of chemotherapeutic drugs
1. Study object
Gastric cancer cell SGC7901 VCR was the same as in example 3 and MKN45 cell was the same as in example 5.
2. Research method
Two cell lines (siSP #1 and siSP # 2) with successful gastric cancer cell lines SGC7901 VCR and MKN45 knockdown of SP1 gene constructed in example 5 were treated with vincristine (MCE, 10. Mu.g/mL) and fluorouracil (MCE, 1. Mu.g/mL), respectively, for 24h, with SGC7901 VCR and MKN45 cells without SP1 gene knockdown as controls. Cells after chemotherapy treatment were collected and apoptosis was detected using Annexin V-FITC apoptosis detection kit (Invitrogen). Fluorescent intensities of Annexin V-FITC and propylene iodide were detected using a Coulter Epics XL-MCL flow cytometer (Beckman) and data analysis was performed using EXPO32 ADC software. Annexin V-FITC-stained positive cells are considered to undergo apoptosis.
3. Conclusion of the study
FIG. 6 shows apoptosis rate of SP1 gene knockdown and control human gastric cancer cells under chemotherapeutic drug treatment conditions. FIG. 6A is a statistical plot of apoptosis rate of human gastric cancer cell SGC7901 VCR with SP1 gene knockdown and control under vincristine treatment conditions, with differences significant (P < 0.01); fig. 6B is a statistical plot of apoptosis rate of the SP1 knock-down and control human gastric cancer cell MKN45 under fluorouracil treatment conditions, with differences significant (P < 0.01). The above results indicate that knocking down the SP1 gene in SGC7901 VCR and MKN45 cells was found to down-regulate SP1 (siSP #1 and siSP # 2) to increase the reactivity to the chemotherapeutic drugs Vincristine (Vincristine) and fluorouracil (Fluorouracil).
Example 7: relation between expression and survival of SP1 gene in different tumor tissues
1. Study object
Based on the relevant data in the TCGA database, the relationship between the expression and survival of SP1 gene in esophageal cancer, brain low-grade glioma, liver cancer, and pancreatic cancer tissues was analyzed.
2. Analysis method
Downloading related data in a TCGA database, and dividing tumor patients with high SP1 expression and low SP1 expression by a best dividing value algorithm; for survival analysis, P-values between groups were calculated using a log rank sum test; and drawing a survival curve by using a Kaplan-Meier method.
3. Conclusion of the experiment
FIG. 7 is a graph showing the overall survival and progression free survival of patients with esophageal cancer (ESCA), low-grade glioma (LGG), liver cancer (LIHC) and pancreatic cancer (PAAD) with SP1 gene under-and over-expression, as analyzed by TCGA data. FIG. 6A is a graph showing analysis of total survival of patients with esophageal cancer, low-grade glioma of the brain, liver cancer and pancreatic cancer with SP1 gene low expression (SP 1 low) and SP1 gene high expression (SP 1 high); FIG. 6B is a graph showing the progression-free survival analysis of patients with esophageal cancer, low-grade glioma, liver cancer and pancreatic cancer, in which the SP1 gene is expressed under and under. The results show that the total survival time and the progression-free survival time of the tumor patients with high expression of the SP1 are shorter than those of the tumor patients with low expression of the SP1, and the SP1 has tumor marker value in esophagus cancer, brain low-grade glioma, liver cancer and pancreatic cancer.
Claims (10)
- Application of the SP1 gene or the coded protein thereof as a target point in preparing a reagent or a kit for diagnosing and/or evaluating the drug resistance of solid tumors; the solid tumor is gastric cancer, intestinal cancer, esophageal cancer, brain low-grade glioma, liver cancer or pancreatic cancer.
- Application of detection reagent of SP1 gene or its coded protein in preparation of reagent or kit for drug resistance diagnosis and/or prognosis evaluation of solid tumor; the solid tumor is gastric cancer, intestinal cancer, esophageal cancer, brain low-grade glioma, liver cancer or pancreatic cancer.
- 3. The use according to claim 1 or 2, wherein the reagent or kit detects transcribed messenger RNA of the SP1 gene or SP1 gene-encoded protein by RT-PCR, real-time quantitative PCR, digital PCR, fluorescent dye method, resonance light scattering method, sequencing or bio mass spectrometry, in situ hybridization, northern blotting, chip, high throughput sequencing platform, immunohistochemistry or enzyme linked immunosorbent method.
- 4. The use according to claim 1 or 2, wherein the reagent or kit contains specific primers for amplifying the SP1 gene, probes hybridizing to the nucleotide sequence of the SP1 gene or antibodies or antibody fragments specifically binding to the SP1 protein.
- 5. The use according to claim 3, wherein the antibody is a monoclonal or polyclonal antibody.
- 6. The use according to any one of claims 1 to 5, wherein the test sample of the reagent or kit is serum, plasma, cells, cell culture supernatant, urine, tissue or tissue lysate.
- Application of SP1 gene or its coded protein as target point in preparing medicine for treating drug-resistant solid tumor; the solid tumor is gastric cancer, intestinal cancer, esophageal cancer, brain low-grade glioma, liver cancer or pancreatic cancer.
- Application of an inhibitor of the expression of an SP1 gene or an inhibitor of the expression of an SP1 gene-encoded protein in preparing a medicament for treating drug-resistant solid tumors; the solid tumor is gastric cancer, intestinal cancer, esophageal cancer, brain low-grade glioma, liver cancer or pancreatic cancer.
- 9. The use of claim 8, wherein the SP1 gene expression inhibitor is selected from the group consisting of a CRISPR gene editing therapeutic agent, an antisense nucleic acid agent, an siRNA agent, or a miRNA agent that blocks the normal transcription or post-transcriptional translation process of an SP1 gene by CRISPR/Cas9 gene editing techniques or RNA interference techniques.
- 10. The use according to claim 8, wherein the inhibitor of SP1 protein expression is selected from the group consisting of protein glycosylation inhibitors, protein phosphorylation inhibitors or neutralizing antibodies affecting post-translational modification of SP1 protein or affecting stability of SP1 protein, and affecting SP1 protein expression level, activity or function.
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