CN114045336B - Application of CGA gene as target spot in preparation of medicine for diagnosing and treating drug-resistant solid tumor - Google Patents
Application of CGA gene as target spot in preparation of medicine for diagnosing and treating drug-resistant solid tumor Download PDFInfo
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- CN114045336B CN114045336B CN202111143449.XA CN202111143449A CN114045336B CN 114045336 B CN114045336 B CN 114045336B CN 202111143449 A CN202111143449 A CN 202111143449A CN 114045336 B CN114045336 B CN 114045336B
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Abstract
The invention discloses application of CGA gene as a target point in preparing a medicine for diagnosing and treating drug-resistant solid tumors, and particularly discloses new discovery of drug resistance of solid tumors based on high expression of CGA in solid tumor tissues, wherein the gene or/and protein is used as the target point for diagnosing and treating drug-resistant solid tumors.
Description
Technical Field
The invention relates to a technology for diagnosing and prognosticating drug resistance of solid tumors, in particular to a novel discovery that glycoprotein hormone alpha polypeptides (CGA, glycoprotein hormones, alpha polypeptide) are highly expressed in solid tumor tissues and cause drug resistance of the solid tumors, and the gene or/and protein is used as a target point for diagnosing and prognosticating drug resistance of the solid tumors.
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 of the invention, the invention provides an application of CGA gene or coded protein thereof as a target in preparing a reagent or a kit for diagnosing and/or prognosis evaluating solid tumor drug resistance.
Furthermore, the scheme of the invention is the application of the CGA gene or the detection reagent of the coded protein thereof in preparing a reagent or a kit for diagnosing and/or prognosis evaluating the drug resistance of the solid tumor.
Further, the above reagents or kits detect messenger RNA transcribed from CGA genes or CGA gene-encoded proteins 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 assay.
Further, the reagent or the kit contains a specific primer for amplifying the CGA gene, a probe hybridized with the nucleotide sequence of the CGA gene or an antibody fragment specifically bound with the CGA protein.
Further, the antibody is a monoclonal antibody or a polyclonal antibody.
Further, the detection 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 CGA gene or the coded protein thereof serving as a target spot in preparing a drug for treating drug-resistant solid tumors.
Furthermore, the scheme of the invention is the application of the CGA gene expression inhibitor or the protein expression inhibitor in preparing medicines for treating drug-resistant solid tumors.
Further, the CGA 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 normal transcription or post-transcriptional translation of a CGA gene by a CRISPR/Cas9 gene editing technique or an RNA interference technique.
Further, the CGA protein expression inhibitor is selected from the group consisting of protein glycosylation inhibitors, protein phosphorylation inhibitors, or neutralizing antibodies that affect CGA protein post-translational modification processes or affect CGA protein stability, and affect CGA protein expression levels, activity, or function.
The invention also provides a construction method of the drug-resistant solid tumor cell research model, and the method provided by the invention comprises the following steps: the CGA gene in the solid tumor cells is knocked out.
In a further embodiment, solid tumor cells are cultured in a medium containing one or more chemotherapeutic agents to construct primary drug-resistant solid tumor cells, and then CGA genes in the primary drug-resistant solid tumor cells are knocked out to obtain drug-resistant solid tumor cells.
Further, the CRISPR/Cas9 technology is adopted to knock out CGA genes in the initial drug-resistant solid tumor cells to obtain the drug-resistant solid tumor cells.
The invention also provides a construction method of the drug-resistant solid tumor animal model, and the method provided by the invention comprises the following steps: and injecting the drug-resistant solid tumor cells constructed by the method into an animal model to construct a drug-resistant solid tumor animal model.
Drawings
FIG. 1 shows a gastric cancer-resistant cell line SGC7901 of example 1 ADR And SGC7901 VCR Results of CGAmRNA expression levels from the control cell line SGC 7901.
FIG. 2 is a gastric cancer-resistant cell line SGC7901 of example 2 ADR And SGC7901 VCR Results of the amount of CGA protein expressed in cells and culture supernatants from the control cell line SGC 7901.
FIG. 3 is a gastric cancer-resistant cell line SGC7901 of example 3 ADR And SGC7901 VCR Immunofluorescence staining of CGA protein expression from control cell line SGC 7901.
FIG. 4 shows the expression changes of CGAmRNA under the induction of sub-lethal doses of chemotherapeutic drug in the chemotherapeutic drug sensitive gastric cancer cells SGC7901 and NCI-N87 of example 4; FIG. 4A is a comparison of CGAmRNA expression levels of SGC7901 and NCI-N87 cells after days 1, 3, 5 and 7 in fluorouracil (1. Mu.g/ml) culture conditions with CGAmRNA expression levels of untreated control cells, and FIG. 4B is a comparison of CGAmRNA expression levels of SGC7901 and NCI-N87 cells after days 1, 3, 5 and 7 in doxorubicin (0.5. Mu.g/ml) culture conditions with CGAmRNA expression levels of untreated control cells.
FIG. 5A is an IHC staining micrograph of CGA protein in Post-chemotherapy gastric cancer tissue (Post) and its paired Pre-chemotherapy gastric cancer tissue (Pre) of a representative 6 gastric cancer patients receiving stable or progressive Post-chemotherapy disease in example 5; FIG. 5B is a statistical plot of IHC scores of CGA expression in post-chemotherapy gastric cancer tissue and paired pre-chemotherapy gastric cancer tissue of 31 gastric cancer patients with stable or progressive disease after chemotherapy; fig. 5C is an IHC staining micrograph of CGA protein in post-chemotherapy gastric cancer tissue and paired pre-chemotherapy gastric cancer tissue of 6 post-chemotherapy partially alleviated gastric cancer patients.
FIG. 6A is an IHC staining micrograph of CGA protein in transplanted tumor tissue after treatment with a control group (Saline) or a chemotherapeutic agent treatment group (Fluoraceil) in mice based on a xenograft (Patient derived xenograft, PDX) model of 4 gastric cancer patients; fig. 6B is a graph of the growth of tumor volumes in the 4 PDX mice receiving chemotherapy and their controls.
FIG. 7A is a statistical plot of the results of Enzyme-linked immunosorbent assay (ELISA) for detecting CGA protein content in plasma samples of human subjects from a Normal control group (Normal), a gastric Non-chemotherapeutic group (Non-chemo), a gastric neoadjuvant chemotherapeutic group (Neoadjuvant chemo) and a gastric palliative chemotherapeutic group (palliative chemo); FIG. 7B is a statistical plot of results of CGA protein content in plasma of patients in the gastric cancer neoadjuvant chemotherapy group divided into two groups, disease Stabilization (SD) and Partial Remission (PR) according to their evaluation of chemotherapeutic efficacy; FIG. 7C is a statistical plot of the results of CGA protein content in plasma samples paired pre-and post-operatively for patients with a neoadjuvant chemotherapy group of gastric cancer; FIG. 7D is a statistical plot of the results of CGA protein content in paired plasma samples of patients in the gastric cancer palliative chemotherapy group prior to chemotherapy and after 4-6 cycles of chemotherapy; FIG. 7E is a statistical plot of results of CGA protein content in plasma of patients with gastric cancer palliative chemotherapy groups divided into two groups, disease Progression (PD) and disease Stabilization (SD) according to their evaluation of chemotherapeutic efficacy; FIG. 7F is a graph showing survival of 64 patients with gastric cancer undergoing chemotherapy and following follow-up, after being divided into CGA low expression and CGA high expression according to median of plasma CGA concentration (304.7 pg/ml).
FIG. 8A shows Oncomine data analysis results, with no statistical difference in CGA expression in gastric cancer tissue and normal gastric mucosal tissue; fig. 8B shows the results of GEPIA data analysis, with no statistical differences in CGA expression in gastric cancer tissue and normal gastric mucosal tissue.
FIG. 9A is SGC7901 ADR And SGC7901 VCR Schematic of CGA gene knockout sites in cells and a DNA sequencing result diagram; FIG. 9B shows a Western Blotting (WB) of SGC7901 ADR And SGC7901 VCR CGA protein expression in two monoclonal cell lines (KO-1 and KO-2) with CGA gene knocked out in cells.
FIG. 10A shows SSGC7901 of CGA genes WT and KO ADR A fitted curve of cell viability versus drug concentration for cells treated with fluorouracil and doxorubicin at different concentrations; FIG. 10B SGC7901 of CGA genes WT and KO VCR A fitted curve of cell viability versus drug concentration for cells treated with fluorouracil and paclitaxel at different concentrations.
FIG. 11A SGC7901 of CGA genes WT and KO ADR A statistical graph of the number of apoptosis after treatment with fluorouracil and doxorubicin; FIG. 11B SGC7901 of CGA genes WT and KO VCR Statistical graphs of the number of apoptosis after treatment with fluorouracil and paclitaxel.
FIG. 12A SGC7901 of CGA genes WT and KO ADR Cell growth curves of cells under fluorouracil and doxorubicin treatment; FIG. 12B SGC7901 of CGA genes WT and KO VCR Cell growth curve of cells under fluorouracil and paclitaxel treatment.
FIGS. 13A and 13B are SGC7901 of CGA genes WT and KO ADR And SGC7901 VCR Tumor growth curve of cells in nude mice under fluorouracil, doxorubicin and paclitaxel treatment conditions; FIGS. 13C and 13D SGC7901 of CGA genes WT and KO ADR And SGC7901 VCR Tumor weight statistics of cells in nude mice under fluorouracil, doxorubicin and paclitaxel treatment conditions; FIGS. 13E and 13F are CGA basesSGC7901 due to WT and KO ADR And SGC7901 VCR Representative micrograph of cells after IHC staining with Ki-67 and clear Caspase-3 protein in nude mice under fluorouracil, doxorubicin and paclitaxel treatment conditions.
FIG. 14A is a schematic representation of a screening strategy for specific miRNAs targeting CGA; FIG. 14B shows miR-17-3p, miR-630, miR-708-3p and miR-761 in gastric cancer drug-resistant cell SGC7901 ADR And SGC7901 VCR Expression in the parent SGC7901 cell thereof; FIG. 14C shows the effect of SGC7901 on gastric cancer resistant cells ADR And SGC7901 VCR After being transfected with the analogs of miR-17-3p, miR-630, miR-708-3p and miR-761, WB detects the expression change of CGA protein in cells.
FIG. 15A is SGC7901 transfected with miR-708-3p and miR-761 mimetic ADR Cell viability under fluorouracil and doxorubicin treatment; FIG. 15B is SGC7901 transfected with miR-708-3p and miR-761 mimetic VCR Cell viability under fluorouracil and paclitaxel treatment.
FIG. 16A is SGC7901 ADR Tumor growth curves after cell neoplasia under 6 different treatment conditions, FIG. 16B is SGC7901 ADR Tumor pictures under 6 different treatment conditions after cell neoplasia, FIG. 16C is SGC7901 ADR Tumor weight statistics at 6 different treatment conditions after cell neoplasia; FIG. 16D is SGC7901 ADR Representative micrograph of Ki-67 and clear Caspase-3 protein IHC staining was performed under 6 different treatment conditions after cell neoplasia.
FIG. 17A is SGC7901 with CGA gene knocked out ADR Cell viability under fluorouracil and doxorubicin treatment after addition of CGA recombinant protein (rCGA); FIG. 17B is SGC7901 with CGA knockdown ADR Cell viability under fluorouracil and doxorubicin treatment following transfection of cells with CGA expression plasmids of WT, N52Q, N78Q or DM.
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.
Glycoprotein hormone alpha polypeptides (glycoprotein hormones, alpha polypeptide, CGA) are common alpha subunits of 4 glycoprotein hormones (human chorionic gonadotrophin, follicle stimulating hormone, luteinizing hormone and thyroid stimulating hormone) in humans. Under normal physiological conditions, human chorionic gonadotrophin is mainly secreted by trophoblast cells of the placenta, and follicle stimulating hormone, luteinizing hormone and thyroid stimulating hormone are mainly secreted by pituitary cells. The 4 glycoprotein hormones are composed of alpha subunit and beta subunit, wherein the beta subunit is encoded by different genes, the alpha subunit is encoded by CGA gene, the CGA gene exists on human chromosome 6, the sequence area is 9.6kbp, 2 transcripts are provided, and 2 protein subtypes are respectively encoded.
The mRNA transcribed from the CGA gene of the invention (abbreviated CGA mRNA) is intended to include the full-length ribonucleotide sequence thereof, or naturally occurring variants, or fragments of full-length sequences and variants, in particular fragments which can be detected and specific sequences determined, more preferably fragments which are distinguishable from other RNA sequences in solid tumor tissue. Preferably at least 7, 8, 9, 10, 11, 12, 15 or 20 consecutive ribonucleotides comprising the full-length ribonucleotide sequence.
The CGA gene-encoded proteins of the invention are intended to include naturally occurring variants of said proteins as well as fragments, in particular immunologically detectable fragments, of said proteins or of said variants. The immunologically detectable fragment preferably comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20 consecutive amino acids of the marker polypeptide. The expression "protein encoded by the CGA gene" includes the complete protein sequence of CGA, as well as the marker polypeptides defined above.
In this application, the term "solid tumor resistance" refers to 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 IC50 (half inhibitory concentration, meaning the concentration of drug required to reduce the number of cells surviving a half after administration) of a chemotherapeutic agent by a factor of more than 2 times after induction or other treatment, as compared to a parent cell that is sensitive to the chemotherapeutic agent (chemosensitivity means that the inhibition rate of the cell growth of the chemotherapeutic agent reaches more than 60% at its normal blood concentration). For clinical solid tumor patients: according to the "efficacy evaluation criteria for solid tumors," version 1.1 (Response Evaluation Criteria in Solid Tumors RECIST Version 1.1.1), patients with neoadjuvant chemotherapy were treated and judged to be disease progression (Progression Diseased, PD) and stable (StableDisease, SD); after treatment, the palliative chemotherapy patients were judged to be disease progression (Progression Diseased, PD).
The invention relates to application of a detection reagent of CGA mRNA or protein in preparation of a reagent or a kit for diagnosing and/or prognosis evaluation of solid tumor drug resistance, wherein the expression level of the CGA mRNA or protein is increased to indicate the solid tumor drug resistance and/or to indicate poor prognosis of gastric cancer.
The term "detection reagent for CGA mRNA" is not to be understood in the present invention as merely a detection reagent for CGA mRNA, but is intended to include the remaining detection reagents known to those skilled in the art that reflect the expression level of CGA mRNA. For example, the amount of expression of CGA mRNA can be indirectly detected by quantitatively detecting cDNA obtained by reverse transcription of CGA mRNA.
The "detection reagent for CGA mRNA" 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 for CGA mRNA 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.
In some embodiments, the quantitative detection agent of CGAmRNA is a probe or primer capable of specifically binding to CGAmRNA or CGA cDNA.
According to one aspect of the invention, the invention also relates to qRT-PCR primers of CGA mRNA, the upstream primers of which are shown in SEQ ID NO:1, the downstream primer is shown as SEQ ID NO: 2. The primer can be used for diagnosis and/or prognosis evaluation of human gastric cancer drug resistance.
The term "antibody" includes polyclonal and monoclonal antibodies,the term "antibody fragment" includes antigen-compound-binding fragments of these antibodies, including Fab, F (ab') 2 Fd, fv, scFv, bispecific antibodies and antibody minimal recognition units, as well as single chain derivatives of these antibodies and fragments, such as scFv-Fc, and the like. The type of antibody may be selected from the group consisting of IgG1, igG2, igG3, igG4, and IgA, igM, igE, igD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric, bifunctional, humanized 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 CGA gene expression inhibitor refers to an agent, a preparation or a drug for blocking the normal transcription or posttranscriptional translation process of the CGA gene based on the CRISPR/Cas9 gene editing technology or the RNA interference technology, such as CRISPR gene editing therapeutic drugs, antisense nucleic acid drugs, siRNA drugs, miRNA drugs and the like.
The CGA 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 CGA protein or the stability of the CGA protein and the expression level, activity or function of the CGA 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.
Gastric cancer is a cancer that occurs in the gastric mucosa, and most gastric cancers belong to adenocarcinoma. In the following examples, stomach cancers not specifically described are gastric adenocarcinoma. However, the research results of the present invention are not limited to gastric cancer, and related applications can be associated with solid tumors including lung cancer, breast cancer, colorectal cancer, liver cancer, ovarian cancer, and prostate cancer according to the conventional knowledge of those skilled in the art.
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.
Wherein the cell lines SGC7901, SGC7901 used in the examples ADR And SGC7901 VCR All are stored in the national key laboratories of tumor biology at the medical university of air force.
SPSS 18.0 software was used for data statistics and analysis; p <0.05 represents a statistical result is significant.
Example 1: real-time quantitative PCR (RT-qPCR) detection of CGAmRNA in gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR Expression differences from the control cell line SGC7901
1. Study object
Gastric adenocarcinoma cell line SGC7901 is from the military medical academy of sciences and is preserved by the national focus laboratory of tumor biology at the medical university of air force army;
SGC7901 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum;
drug-resistant gastric cancer cell SGC7901 ADR And SGC7901 VCR The cell is established by adopting an intermittent induction method, which is formed by respectively exposing SGC7901 parent cells to a lethal dose of doxorubicin and vincristine culture medium for a short time and inducing the cells generation by generation, wherein the concentration of the doxorubicin is 2.5 mug/ml, and the concentration of the vincristine is gradually increased from 1 mug/ml to 5 mug/ml; specifically, SGC7901 ADR The cells were cultured for a long period of time using DMEM medium SGC7901 containing 0.05. Mu.g/ml doxorubicin and 10% fetal bovine serum VCR The long-term culture of cells comprises culturing with DMEM medium containing 0.5 μg/ml vincristine and 10% foetal calf serum at 37deg.C and 5% (v/v) CO 2 After the cells are cultured to logarithmic phase, the IC50 of the cultured cells is increased by more than 2 times compared with that of chemotherapy sensitive cells (namely parent cells), and the cells are collected to obtain drug-resistant gastric cancer cells SGC7901 ADR And SGC7901 VCR Subsequent experiments were performed. Established SGC7901 ADR And SGC7901 VCR Cell p-fluorouracil, doxorubicinCross-resistance of chemotherapeutic agents such as paclitaxel.
2. Experimental method
Detection of CGA Gene at SGC7901 Using RT-qPCR ADR And SGC7901 VCR And SGC7901 cells, in the following steps:
after each cell was cultured for 24 hours, total RNA was extracted from the cells using the RNA isolation reagent TRIzol, and the concentration and quality of RNA was measured using an ultraviolet spectrophotometer;
cDNA was obtained using Applied Biological Materials Inc.5×all-In-One RT Master mix reverse transcription RNA, reverse transcription System: RNA 1. Mu.g, 5 XAll-In-One RT MasterMix 4. Mu.l, ddH 2 O was added to the reaction volume to 20. Mu.l; reaction conditions: incubating at 25 ℃ for 10min, incubating at 42 ℃ for 15min, and inactivating enzyme at 85 ℃ for 5min;
Using TaKaRa TB GreenPremix Ex Taq TM (TliRNaseH Plus), RT-qPCR detection is carried out on Bulk by using a reverse rate product as a template through a TB Green chimeric fluorescence method, and the reaction system is as follows: TB Green Premix Ex Taq (2X) (TliRNaseH Plus), bulk 12.5. Mu.l, upstream primer 1. Mu.l, downstream primer 1. Mu.l, cDNA 1. Mu.l, ddH 2 O was added to the reaction volume to 25. Mu.l; reaction conditions: reacting at 95 ℃ for 30s, and circulating at 95 ℃ for 5s for 40 times; 60 ℃ for 30s; collecting fluorescence at 60 ℃; dissolution profile, increasing by 0.5 ℃ per cycle from 60 ℃ to 95 ℃; the primer sequences of the PCR reactions are shown in Table 1; by 2 -△△CT The quantitative results were calculated by the method, and the expression amounts of the mRNAs in the respective cell lines were statistically analyzed.
TABLE 1 RT-qPCR primer sequences
3. Conclusion of the experiment
FIG. 1 shows a gastric cancer drug-resistant cell line SGC7901 ADR And SGC7901 VCR Results of CGAmRNA expression levels with the control cell line SGC 7901; as can be seen from FIG. 1, CGAmRNA was found to be useful in gastric cancer resistant cell line SGC7901 ADR And SGC7901 VCR The expression in the cell line was elevated compared with that of the control cell line SGC7901, and the difference was significant (P<0.05 Indicating that CGAmRNA expression levels are associated with gastric cancer multi-drug resistance.
Example 2: WB detection CGA protein in gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR Expression differences from the control cell line SGC7901
1. Study object
Gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR Culture of SGC7901, a control cell line, was performed as in example 1.
2. Experimental method
Detection of CGA proteins at SGC7901 Using WB ADR And SGC7901 VCR And SGC7901 cells, in the following steps:
after each cell is normally cultured for 24 hours, replacing a DMEM culture medium without fetal calf serum for continuous culture for 24 hours, and collecting culture supernatants and cells respectively;
centrifugation at 4000 Xg for 1h at 4℃using Millipore ultrafiltration tube to collect secreted proteins of the culture supernatant, extraction of total cellular proteins using RIPA cell lysate, BCA assay to determine the concentration and quality of supernatant and cellular sample proteins;
electrophoresis was performed using a standard electrophoresis apparatus of Bio-Rad, and a fast transfer membrane was performed using a Bio-Rad; after blocking the PVDF membrane, the primary antibody (rabbit anti-human CGA mab, abcam,1:1000; mouse anti-human alpha-Tubulin mab, sigma-Aldrich, 1:2000) was incubated overnight at 4 ℃. Washing with PBS for 5min, repeating for 3 times, and incubating with secondary antibody (HRP-labeled donkey anti-rabbit, sheep anti-mouse IgG, GE Healthcare Life Sciences, 1:5000) at room temperature for 1h; the washing with PBS was repeated 3 times for 5min, and the gray scale values of the protein bands were determined using an ECL chemiluminescent solution and a Bio-Rad ChemiDox XRS gel imaging system.
3. Conclusion of the experiment
FIG. 2 shows a gastric cancer drug-resistant cell line SGC7901 ADR And SGC7901 VCR Results of CGA protein expression levels in cells and culture supernatants from control cell line SGC 7901; as can be seen from FIG. 2, gastric cancer drug-resistant cell line SGC7901 ADR And SGC7901 VCR The intracellular and secretion of CGA protein in culture supernatant is higher than that of SGC7901 of control cell strain, which shows that the expression level of CGA protein is similar to that of gastric cancer multidrug resistanceAnd (3) closing.
Example 3: immunofluorescence (IF) detection of CGA protein in gastric cancer resistant cell line SGC7901 ADR And SGC7901 VCR Differences in expression and subcellular level localization from control cell line SGC7901
1. Study object
Gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR Culture of SGC7901, a control cell line, was performed as in example 1.
2. Experimental method
Detection of CGA proteins at SGC7901 Using IF ADR 、SGC7901 VCR And SGC7901 cells, the specific steps are as follows:
after each cell is inoculated to a chamber glass slide for conventional culture for 24 hours, 4% paraformaldehyde is added to fix the cell, 0.5% Triton is added, and the cell is allowed to stand at room temperature for 15min to permeabilize the cell; dripping goat serum sealing liquid, and standing at room temperature for 30min; primary antibody (rabbit anti-human CGA mab, abcam, 1:1000) was incubated overnight at 4 ℃. Washing with PBS for 5min, 3 times, and incubating the secondary antibody (Alexa Fluor Plus 594 labeled donkey anti-rabbit IgG, thermo Fisher Scientific, 1:200) at room temperature in the dark for 1h. Washing with PBS for 5min, repeating for 3 times, and nonspecifically staining cell nuclei with DAPI for 15min; fluorescence expression was observed after sealing using a Nikon A1 confocal microscope.
3. Conclusion of the experiment
FIG. 3 shows a gastric cancer drug-resistant cell line SGC7901 ADR And SGC7901 VCR Immunofluorescence results of CGA protein expression from control cell line SGC 7901; as can be seen from FIG. 3, the gastric cancer drug-resistant cell line SGC7901 ADR And SGC7901 VCR The CGA protein content in the cells is higher than that of the cell strain SGC7901 in the control group, and is mainly expressed in SGC7901 ADR And SGC7901 VCR The cytoplasm and cell membrane of the cell indicate that the CGA protein is highly expressed in gastric cancer multidrug resistant cells.
Example 4: RT-qPCR detection of expression change of CGAmRNA of gastric cancer cell lines SGC7901 and NCI-N87 under induction condition of sublethal dose of chemotherapeutic drugs
1. Study object
Culture of gastric adenocarcinoma cells SGC7901 was the same as in example 1; gastric adenocarcinoma cells NCI-N87 were purchased from american type culture collection (American type culture collection, ATCC) under the same culture conditions as SGC7901 cells; both of these cells are sensitive to the chemotherapeutics fluorouracil and doxorubicin;
2. experimental method
The expression changes of the CGA 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% mortality after 24h of drug treatment) of the chemotherapeutic drug.
Specifically, RT-qPCR was used to detect the expression levels of CGA gene on days 1, 3, 5, 7 after receiving sublethal doses of fluorouracil (1. Mu.g/ml) and doxorubicin (0.5. Mu.g/ml) in SGC7901 and NCI-N87 cells, following the procedure of example 1.
3. Conclusion of the experiment
FIG. 4 shows the expression changes of CGAmRNA of chemotherapeutic sensitive gastric cancer cells SGC7901 and NCI-N87 under the induction condition of sub-lethal dose of chemotherapeutic; FIG. 4A is a comparison of CGAmRNA expression levels of SGC7901 and NCI-N87 cells after days 1, 3, 5 and 7 in fluorouracil (1. Mu.g/ml) culture conditions with CGAmRNA expression levels of untreated control cells, and FIG. 4B is a comparison of CGAmRNA expression levels of SGC7901 and NCI-N87 cells after days 1, 3, 5 and 7 in doxorubicin (0.5. Mu.g/ml) culture conditions with CGAmRNA expression levels of untreated control cells. As can be seen from fig. 4, the chemotherapeutic agent can induce CGA gene expression, and the increase in cells with longer survival time under the chemotherapeutic condition is more obvious, which indicates that the expression level of CGA can reflect the drug-resistant state of gastric cancer cells.
Example 5: immunohistochemistry (IHC) for detecting expression of CGA protein in stomach cancer tissues paired before and after chemotherapy of stomach cancer patients receiving chemotherapy
1. Study object
Collecting 37 paraffin embedded tissue samples of gastric cancer tissues of patients with gastric cancer subjected to neoadjuvant chemotherapy before and after chemotherapy; the pre-chemotherapy sample is taken from a gastroscope biopsy, the post-chemotherapy sample is taken from a gastric cancer operation, and the patient is not subjected to radiotherapy or other treatments prior to the biopsy or operation. The paraffin embedded tissue samples used in this example were all from the air force medical university national key laboratory tissue sample library of tumor biology; each patient signed an informed consent prior to sampling according to the regimen prescribed by the ethical review board. General conditions and pathological information of 37 gastric cancer patients are collected in Table 2, wherein 31 patients are resistant to chemotherapy, and 6 patients are sensitive to chemotherapy.
TABLE 2 general and pathological information of 37 gastric cancer patients
1 Chemotherapy regimen: FOLFOX, fluorouracil, calcium folinate, oxaliplatin; EOX, epirubicin, oxaliplatin, capecitabine; DCF, docetaxel, cisplatin, fluorouracil; ECF, epirubicin, cisplatin, fluorouracil; XELOX, capecitabine, oxaliplatin; SOX, tegafur, oxaliplatin; FOLFIRI, fluorouracil, calcium folinate, irinotecan; salt, fluorouracil, calcium folinate, oxaliplatin and docetaxel.
2 Evaluation of curative effect: PR, partial relief; SD, stable disease; PD, disease progression.
2. Definition of index of sensitivity to chemotherapy and chemotherapy drug resistance of gastric cancer patients in study
Chemosensitivity: refers to the evaluation of efficacy of a neoadjuvant chemotherapy regimen prior to receiving surgery as Complete Remission (CR) or PR, according to the solid tumor efficacy evaluation criteria (Response Evaluation Criteria in Solid Tumors-RECIST) version 1.1.
Chemotherapy resistance: refers to the evaluation of efficacy of a neoadjuvant chemotherapy regimen for gastric cancer patients prior to receiving surgery as SD or PD according to RECIST version 1.1.
3. Experimental method
The IHC is adopted to detect the expression level of CGA protein in gastric cancer tissues before and after chemotherapy of 37 gastric cancer patients, and the specific steps are as follows:
Placing the tissue slice in a 60 ℃ incubator, baking for about 1h, dewaxing in xylene for 10min, repeating for 3 times, and sequentially placing in 99.9%, 96% and 70% ethanol for 5min for dehydration, repeating for 3 times;
boiling the slices in sodium citrate antigen retrieval solution for 15min, and placing the slices in 3%H 2 O 2 Dripping the sealing liquid for incubation for 30min at room temperature for 15 min; primary antibody (rabbit anti-human CGA mab, abcam, 1:200) was incubated overnight at 4 ℃; washing with PBS for 5min, repeating for 3 times, and incubating the secondary antibody (biotin-labeled goat anti-rabbit IgG polymer, zhonghua gold bridge) for 1h at room temperature; washing with PBS, DAB color development, hematoxylin staining of cell nuclei. Differentiating 1% hydrochloric acid alcohol, returning blue with 0.1% ammonia water, sequentially dehydrating slices in 70%, 96% and 99.9% ethanol, and sealing with neutral resin;
semi-quantitative scoring of immunohistochemical reactions was performed using immune response integration, staining intensity was rated as 4: no positive cells were counted for 0 score, weak positive for 1 score, medium positive for 2 score and strong positive for 3 score. IHC score = Σ (staining intensity x percentage of positive cells).
4. Conclusion of the experiment
FIG. 5A is an IHC staining micrograph of CGA protein in cancer tissue after gastric cancer chemotherapy (Post) and paired Pre-chemotherapy cancer tissue (Pre) in a representative 6 cases of chemotherapy-resistant gastric cancer patients (Patent 1-6), showing that CGA protein is expressed negatively or weakly positively in cancer tissue before chemotherapy and CGA protein is expressed positively or strongly positively in cancer tissue after chemotherapy. FIG. 5B is a statistical plot of IHC scores of CGA expression in post-and pre-chemotherapy gastric cancer tissues of 31 chemotherapy-resistant gastric cancer patients, showing that 70.96% (22/31) of the 31 post-chemotherapy gastric cancer tissue samples were stained CGA positive; further statistical analysis was performed using a non-parametric rank sum test, and it was found that CGA protein was expressed at elevated levels in cancer tissues after gastric cancer chemotherapy compared to their paired pre-chemotherapy cancer tissues, with significant differences (P < 0.05). FIG. 5C is an IHC staining micrograph of CGA protein in stomach cancer tissues after and before chemotherapy of 6 patients with chemotherapy-sensitive stomach cancer (Patent 7-12), and the results show that the CGA protein has no significant difference in expression in stomach cancer tissues before and after chemotherapy, and are negative. As shown in fig. 5, the CGA protein is significantly increased in the stomach cancer tissues after chemotherapy of the patients with drug-resistant stomach cancer and has significant expression difference in the stomach cancer tissues before and after chemotherapy, and is negatively expressed in the stomach cancer tissues before and after chemotherapy of the patients with chemotherapy-sensitive stomach cancer, which indicates that the CGA protein has significant correlation with the occurrence of gastric cancer drug resistance.
Example 6: IHC detects the expression of CGA protein in tumor tissue of xenograft (Patient derived xenograft, PDX) model derived from gastric cancer patient receiving chemotherapy or control treatment
1. Study object
The stomach cancer tissue is obtained from a stomach cancer patient who is subjected to stomach cancer excision by a first affiliated hospital of the medical university of air force; according to the system specified by the ethical examination committee, the patients sign an informed consent before sampling; after the gastric cancer tissue is resected in the operation, the gastric cancer tissue is immediately put into Hank's balancing solution for transportation;
the PDX model is carried out within 4 hours after gastric cancer tissue is isolated, and the specific steps are as follows:
selecting sterile gastric cancer tissue with good tumor activity, and cutting the tissue into about 3mm 3 Is mixed with Matrigel, and each block is inoculated to 3-5 male NOD-Prkdc respectively em1IDMO -Il2rg em2IDMO (NPI) mice, post-operatively kept in a specific pathogen-free (Specific pathogen free, SPF) environment, designated P0 PDX; body weight of mice was measured weekly, major and minor diameters of tumors were measured weekly after tumorigenesis and tumor volumes were calculated (tumor volume=0.52×major diameter×minor diameter 2 ) The method comprises the steps of carrying out a first treatment on the surface of the When the P0 generation PDX tumor grows to 500mm 3 When the transplanting is carried out, the transplanting method is the same as the previous method, and the P1 generation PDX is marked after the tumor formation. The PDX mice with stable genetic characteristics of the generation P2, the generation P3 and the generation P4 sequentially obtained by the method can be used for subsequent experiments. General and pathological information of 4 gastric cancer patients were collected and are shown in Table 3.
General conditions and pathological information of 3 4 gastric cancer patients
2. Experimental method
The P2-P4 generation gastric cancer PDX mice from the same patient tissue are divided into an experimental group and a control group (3-5 of each group), wherein the experimental group receives an antimetabolite chemotherapeutic drug Fluorouracil (60 mg/kg) for intraperitoneal injection (3 times per week), the control group receives an equal volume of physiological saline for intraperitoneal injection (3 times per week), and tumor volumes of the experimental group and the control group are recorded. After 3-4 weeks, mice are sacrificed according to the theoretical requirements of experimental animals, tumor tissues are fixed, embedded and sectioned, IHC staining is carried out, and the expression of CGA protein is observed. IHC is performed in the same manner as in example 5.
3. Conclusion of the experiment
FIG. 6A is a IHC staining micrograph of CGA protein in transplanted tumor tissue of mice in the treated group and the control group corresponding to PDX1-4, showing that the expression level of CGA protein in the stomach cancer tissue is increased to a different degree after the stomach cancer tissue is treated by the chemotherapeutic drug compared with the tissue in the control treated group. Fig. 6B is a graph of tumor volumes of 4 PDX mice receiving chemotherapy and their controls, reflecting the responsiveness of patient-derived tumor tissue to chemotherapeutic agents: the experimental group of PDX1 shows that the tumor grows slowly after treatment, and the experimental group is sensitive to fluorouracil; the tumor volume remained elevated after treatment in the PDX2-4 experimental group, indicating that it was resistant to fluorouracil. Analysis of the expression of CGA protein in fig. 6A versus the tumor growth curve of fig. 6B revealed that: after receiving chemotherapy, tumors with insignificant CGA protein expression are sensitive to the chemotherapeutic agent, whereas tumors with significant CGA protein expression are resistant to the chemotherapeutic agent. The above results demonstrate that the change in the expression level of CGA protein has a significant correlation with the occurrence of gastric cancer resistance.
Example 7: ELISA (enzyme-Linked immuno sorbent assay) for detecting content of CGA protein in serum of different people and analyzing diagnostic value of CGA on gastric cancer resistance
1. Study object
Plasma samples from 41 cases of gastric cancer patients receiving neoadjuvant chemotherapy (neoadjuvant chemotherapy group Neoadjuvant chemo), 56 cases of gastric cancer patients receiving palliative chemotherapy (palliative chemotherapy group palliative chemotherapy), 42 cases of gastric cancer patients not receiving chemotherapy (Non-chemotherapy group Non-chemo) and 57 cases of Normal persons (Normal control group Normal) in the tissue sample library of the key laboratory in the tumor biology country of the air force medical university were collected. Wherein, the chemotherapy scheme of the patients in the new auxiliary chemotherapy group and the palliative chemotherapy group comprises fluorouracil, taxol, cisplatin and doxorubicin which are used singly or in combination, the plasma of the patients in the non-chemotherapeutic group is collected when the patients are initially diagnosed as gastric cancer and do not receive any radiotherapy and chemotherapy, and the serum of the normal people is the serum of the healthy people which are confirmed to be without upper digestive tumor by gastroscopy and biopsy cases. Each patient signed an informed consent prior to sampling according to the regimen prescribed by the ethical review board. The general condition of 139 gastric cancer patients was collected and shown in tables 4 to 6.
TABLE 4 general and pathological information of 41 patients with gastric cancer receiving neoadjuvant chemotherapy
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1 Chemotherapy regimen: DS, docetaxel, tegafur; DF, cisplatin, fluorouracil; DOX, docetaxel, oxaliplatin, capecitabine.
2 Patient survival was stopped until the end of the follow-up of the study.
TABLE 5 general and pathological information of 56 patients with gastric cancer receiving palliative chemotherapy
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1 Chemotherapy regimen: SP, tegafur, cisplatin; xeiri, capecitabine, irinotecan; EOX, epirubicin, oxaliplatin, capecitabine.
TABLE 6 general and pathological information of 42 patients without chemotherapy gastric cancer
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2. Experimental method
The content of CGA protein in plasma samples of gastric cancer chemotherapeutic group, non-chemotherapeutic group and normal control group was detected by ELISA using CGAELISA kit (product number: NBP 2-75262) from Novus Biologicals Co., U.S.A., specifically comprising the following steps:
5000pg protein standard is dissolved in 1ml sample diluent, and the standard is prepared into 5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 0pg/ml concentration gradients by a double ratio dilution method; plasma and sample dilutions were mixed at 1:2, diluting in proportion; adding standard substances with various concentration gradients and diluted plasma samples into sample application holes of a 96-hole ELISA plate, wherein the sample application amount is 100 mu l/hole, placing the plate at 37 ℃ for incubation for 90min after film sealing, discarding liquid in the 96-hole plate, adding 100 mu l of biotin-labeled CGA antibody diluent (dilution ratio 1:1000), placing the plate at 37 ℃ for incubation for 60min after film sealing, discarding liquid in the 96-hole plate, and washing 3 times by using washing liquid. 100 μl of horseradish peroxide working solution is added into each well, the membrane is closed, the mixture is placed at 37 ℃ for incubation for 30min, the liquid in the 96-well plate is discarded, and the mixture is washed 3 times with washing liquid. Adding 90 μl of substrate working solution, sealing the film, incubating at 37deg.C for 15min, adding 50 μl of stop working solution, and detecting absorbance (OD value) at 450nm with an enzyme-labeled instrument; and (3) carrying out linear regression analysis on different concentration gradient standards and corresponding OD values, fitting a function equation, substituting the corresponding OD values of the plasma sample into the equation, and calculating the concentration of the CGA in the sample.
3. Conclusion of the experiment
Detection of tumor markers in blood is currently the most commonly used method for clinical early diagnosis of tumors, and the results of the detection of CGA protein content in plasma samples of gastric cancer chemotherapeutic group, non-chemotherapeutic group and normal control group by ELISA technique in this example are shown in fig. 7A: the expression level of CGA in the gastric cancer neoadjuvant chemotherapy group (Neoadjuvant chemo) and the palliative chemotherapy group (palliative chemotherapy) is higher than that in the Non-chemotherapy group (Non-chemo) and the Normal control group (Normal), and the difference is significant (P < 0.05); there was no significant difference in mean plasma CGA concentrations between the non-receiving chemotherapy group and the normal control group; as shown in fig. 7B, among the neoadjuvant chemotherapy group patients, patients with efficacy assessment of SD had plasma CGA levels higher than those with efficacy assessment of PR, with differences significant (P < 0.01); as shown in fig. 7C, CGA protein levels were significantly different in plasma paired pre-and post-operation from 15 patients receiving the neoadjuvant chemotherapy group (P < 0.05) after surgery than before surgery. As shown in fig. 7D, CGA protein levels were significantly different in plasma of the preoperative and postoperative pairings of 46 patients receiving palliative chemotherapy after chemotherapy than before chemotherapy (P < 0.01); as shown in fig. 7E, among patients receiving palliative chemotherapy, patients with efficacy assessed as PD had higher levels of CGA protein than patients with efficacy assessed as SD, with differences significant (P < 0.05); as shown in fig. 7F, 64 patients receiving chemotherapy and following the study were divided into two groups according to median CGA protein content in plasma (304.7 pg/ml): CGA high expression group (CGAHigh) and CGA low expression group (CGALow); the Log-rank test shows that the survival time of the patients with high expression of plasma CGA is shorter than that of the patients with low expression of CGA. The results show that the CGA protein content in the blood plasma can reflect the reactivity of a gastric cancer patient to chemotherapy and can be used as an index for predicting the survival time of the patient after receiving the chemotherapy.
Example 8:
based on the studies of the above examples, the inventors further analyzed the expression of CGA gene in gastric cancer and normal tissues based on the relevant data in Oncomine and GEPIA databases
1. Study object
The Oncomine database is the largest cancer gene chip database and integrated data mining platform worldwide at present, integrates RNA and DNA-seq data from a gene expression database (Gene Expression Omnibus, GEO), a tumor genome map (The Cancer Genome Atlas, TCGA), published literature and the like, and can be used for analyzing gene expression differences. The gene expression profiling data interaction analysis (Gene Expression Profiling Interactive Analysis, GEPIA) database is a visual cancer big data analysis platform established on the basis of two transcriptome databases, namely TCGA and genotype-tissue expression (the genetype-tissue expression, GTEx). The inventor analyzes the expression difference of the CGA gene in normal tissues beside gastric cancer and gastric cancer by mining relevant information in two databases of Oncomine and GEPIA so as to further determine whether the CGA can be used as a specific marker of gastric cancer drug resistance.
2. Analysis method
In the Oncomine database (https:// www.oncomine.org /), gastric cancer tissue was compared with a plurality of data sets of normal gastric mucosal tissue, and the expression level of CGA gene in each data set was analyzed. To reduce the error rate, the results were further analyzed with P <0.0001, fold Change >2, and top 10% of the gene rank as screening thresholds. In the GEPIA database (http:// GEPIA. Cancer-pku. Cn /), the differential expression data of CGA in gastric cancer and normal gastric mucosal tissues were retrieved using "CGA" and "STAD" as keywords.
3. Conclusion of analysis
FIG. 8A is a data analysis of CGA mRNA expression in gastric cancer tissue samples and normal gastric mucosal tissue samples in 6 different sources of DNA or RNA-seq data set in Oncomine database, showing that there was no statistical difference in CGA gene expression in gastric cancer and normal gastric mucosal tissue in 6 data. FIG. 8B is a dotted plot of CGAmRNA expression in 408 gastric cancer tissues and 211 normal gastric mucosal tissues in the GEPIA database, indicating that there is no statistical difference in CGA gene expression in gastric cancer tissues and paracancerous normal tissues. As can be seen from fig. 8, there is no difference in the expression of CGA gene in normal stomach tissue and stomach cancer tissue, and in combination with other examples of the present invention, it is shown that CGA is a specific marker for gastric cancer resistance, not a marker for gastric cancer occurrence.
Example 9: construction of cell lines stably knocking out the CGA Gene
1. Study object
Drug-resistant gastric cancer cell SGC7901 ADR And SGC7901 VCR Cell culture was as in example 1.
2. Experimental method
Respectively uses gastric cancer drug-resistant cells SGC7901 ADR And SGC7901 VCR As target cells, CRISPR/Cas9 technology was used to construct cell lines that stably knocked out CGA genes. The specific experimental steps are as follows:
(1) The synthesis of sgrnas was designed. The sgRNA capable of targeting the CGA gene was designed and selected through the sgRNA design website (https:// crispr. Dbcls. Jp), and the nucleotide sequence was shown in Table 7 with the non-targeting sgRNA sequence as a control;
(2) A Cas9 single vector plasmid was constructed. Human codon optimized Cas9 and a sgRNA vector gRNA_GFP-T1 (purchased from Addgene, cat. No. 41819) for expressing recognition GFP are adopted, TOYOBO high-fidelity high-efficiency high-speed PCR enzyme KOD-Plus-Neo kit (cat. No. KOD-401) is adopted, and the sgRNA 19bpDNA for recognizing the CGA gene is inserted into the sgRNA expression vector by using a QuickChangePCR method; the QuickChangePCR reaction system is as follows: 10X PCR Buffer for KOD-Plus-Neo 5. Mu.l, 2mM dNTPs 5. Mu.l, 25mM MgSO 4 Mu.l of each of the upstream and downstream primers (10. Mu.M) 1.5. Mu.l, 50ng of gRNA-GFP-T1 plasmid template, KOD-Plus-Neo 1. Mu.l, ddH 2 O was added to the reaction volume to 50. Mu.l; the QuickChangePCR reaction conditions were: reacting at 94 ℃ for 2min; reacting at 98 ℃ for 10s, at 68 ℃ for 2min, and 22 cycles;
(3) Transfecting the target cell: simultaneous transfection of SGC7901 with 1 μg of sgRNA expression plasmid, 3 μg of Cas9 expression plasmid and 1 μg of EGFP (p-EGFP-C1) expression plasmid ADR And SGC7901 VCR After 3 days, the cells are separated into single cells positive for GFP expression by using a flow cytometry, the single cells are cultured for cloning and growing, and whether gene knockout is successful or not is verified by DNA sequencing and WB after PCR amplification.
Detection of CGA proteins at SGC7901 Using WB ADR And SGC7901 VCR Expression in cells knocked out of the CGA gene was as follows: extracting total cell proteins by using RIPA cell lysate, and determining the protein concentration of a cell sample by a BCA method; standard electrophoresis suit using Bio-Rad Performing electrophoresis, and transferring films by using a Bio-Rad rapid film transfer instrument; after blocking the PVDF membrane, primary antibodies (rabbit anti-human CGA mab, purchased from Abcam,1:1000; mouse anti-human alpha-Tubulin mab, purchased from Sigma-Aldrich, 1:2000) were incubated overnight at 4 ℃. Washing with PBS for 5min, repeating for 3 times, and incubating with secondary antibody (HRP-labeled donkey anti-rabbit, sheep anti-mouse IgG, purchased from GE Healthcare Life Sciences, 1:5000) at room temperature for 1h; the washing with PBS was repeated 3 times for 5min, and the gray scale value of the protein bands was determined by Bio-Rad Chemidox XRS gel imaging system using ECL chemiluminescent solution.
TABLE 7 sgRNA sequences targeting CGA Gene
3. Conclusion of the experiment
FIG. 9A is SGC7901 ADR And SGC7901 VCR The CGA gene knockout site schematic and the DNA sequencing result diagram in the cell show that in the exon 3 of the CGA gene, the sgRNA induces Cas9 protein to be cleaved and then repaired in a non-homologous recombination mode, resulting in CGA gene defect. FIG. 9B shows WB verified SGC7901 ADR And SGC7901 VCR CGA protein expression in two monoclonal cell lines (KO-1 and KO-2) with CGA gene knockdown in cells, indicating that sgRNA successfully induced CGA gene cleavage and defective repair, resulting in loss of CGA protein expression. Through result identification, screening an optimal target point: sgRNA #1 target activity was highest.
Example 10: knockout of CGA Gene on gastric cancer resistant cell line SGC7901 ADR And SGC7901 VCR Influence of cell viability under different treatment with therapeutic drugs
1. Study object
Construction of a successful gastric cancer resistant cell line SGC7901 Using example 9 ADR And SGC7901 VCR Two monoclonal cell lines (KO-1 and KO-2) from which CGA gene was knocked out were subjected to cell proliferation-toxicity experiments to obtain multi-drug resistant SGC7901 without CGA gene knockdown ADR And SGC7901 VCR Cells (WT) served as controls.
2. Experimental method
SGC7901 of CGA genes WT, KO-1, KO-2 ADR And SGC7901 VCR Expanding and culturing cells until the cells are in good condition, digesting each cell with pancreatin, and regulating the concentration of each cell to 5×10 with complete culture medium 4 Uniformly adding the cell suspension into a 96-well plate at a volume of 100 mu l/ml, and culturing at 37 ℃ overnight; after cell attachment, each well was replaced with a corresponding complete medium containing Fluorouracil (Fluorouracil), doxorubicin (Adriamycin) or Paclitaxel (Paclitaxel) in a gradient dilution, 100 μl per well; adding corresponding drug-containing culture mediums with different concentrations into the cell-free holes to serve as blank control; after the end of the drug treatment, 10. Mu.l of CCK-8 solution was added to each well and the incubation was continued at 37℃for 2 hours, absorbance was measured at 450nm, and the experimental results were expressed as cell viability: cell viability (%) = (drug-treated group-blank)/(non-drug-treated group-blank) ×100%.
3. Conclusion of the experiment
FIG. 10A SGC7901 of CGA genes WT and KO ADR A fitted curve of cell survival rate and drug concentration of cells treated by fluorouracil and doxorubicin with different concentrations shows that after CGA gene knockout, gastric cancer drug-resistant cell strain SGC7901 ADR IC50 for fluorouracil and doxorubicin was significantly reduced. FIG. 10B SGC7901 of CGA genes WT and KO VCR A fitted curve of cell survival rate and drug concentration of cells treated by fluorouracil and taxol at different concentrations shows that after CGA gene knockout, gastric cancer drug-resistant cell strain SGC7901 VCR IC50 for fluorouracil and paclitaxel is significantly reduced. As can be seen from fig. 10, CGA gene knockout can lead to a significant decrease in drug resistance of gastric cancer resistant cells.
Example 11: knockout of CGA Gene on gastric cancer resistant cell line SGC7901 ADR And SGC7901 VCR Influence of apoptosis under treatment with chemotherapeutic drugs
1. Study object
Construction of a successful gastric cancer resistant cell line SGC7901 Using example 9 ADR And SGC7901 VCR Two monoclonal cell strains KO-1 and KO-2 of the knock-out CGA gene are subjected to apoptosis detection under the condition of a certain chemotherapeutic drug concentration so as to not knock out CGA gene multidrugDrug resistant WGSGC 7901 ADR And SGC7901 VCR Cells served as controls.
2. Experimental method
SGC7901 of CGA genes WT, KO-1, KO-2 ADR And SGC7901 VCR Expanding the cells to obtain good cell state, digesting each cell with pancreatin, preparing single cell suspension, inoculating into 6-well plate, and cell number per well being about 2×10 5 A plurality of; after the cells are attached, fluorouracil, doxorubicin or paclitaxel with the concentration of 10 mug/ml is added to treat each group of cells; after the drug treatment is finished, preparing the cells into single cell suspension; after washing with PBS pre-chilled to 4℃the cells were resuspended in 200. Mu.l of binding buffer (10 mM HEPES/NaOH, pH 7.4;140mMNaCl;2.5mM CaCl) 2 ) Mu.l of Annexin V-FITC and 5. Mu.l of PI staining solution were added, and after mixing, incubated at 37℃for 30min in the absence of light. 300 μl of binding buffer was added and FITC fluorescence was detected using a flow cytometer at 515nm excitation light wavelength and PI fluorescence was detected at excitation light wavelengths greater than 560 nm.
3. Conclusion of the experiment
FIG. 11A SGC7901 of CGA genes WT and KO ADR Statistical graphs of apoptosis data after treatment of fluorouracil and doxorubicin show that fluorouracil and doxorubicin more easily induce SGC7901 after CGA gene knockout ADR Is a cell apoptosis of (a) a cell. FIG. 11B SGC7901 of CGA genes WT and KO VCR Apoptosis data statistics graph of fluorouracil and paclitaxel cells shows that fluorouracil and paclitaxel can induce SGC7901 after CGA gene knockout more easily VCR Is a cell apoptosis of (a) a cell. As shown in fig. 11, CGA gene knockout can cause a significant decrease in drug resistance of gastric cancer resistant cells, which are prone to chemotherapy-induced apoptosis.
Example 12: knockout of CGA Gene on gastric cancer resistant cell line SGC7901 ADR And SGC7901 VCR Influence of cell proliferation Capacity under treatment with chemotherapeutic drug
1. Study object
Construction of a successful gastric cancer resistant cell line SGC7901 Using example 9 ADR And SGC7901 VCR Knocking out two monoclonal cell strains KO-1 and KO-2 of CGA gene, concentrating in a certain chemotherapeutic medicineCell proliferation potency assay with multidrug resistant WT SGC7901 ADR And SGC7901 VCR Cells served as controls.
2. Experimental method
SGC7901 of CGA genes WT, KO-1, KO-2 ADR And SGC7901 VCR Expanding and culturing cells until the cells are in good condition, digesting each cell with pancreatin, and regulating the concentration of each cell to 5×10 with complete culture medium 4 Uniformly adding the cell suspension into a 96-well plate at a volume of 100 mu l/ml, and culturing at 37 ℃ overnight; after cell attachment, each well was replaced with complete medium containing 10. Mu.g/ml fluorouracil, doxorubicin or paclitaxel, respectively, 100. Mu.l per well; adding corresponding drug-containing culture mediums with different concentrations into the cell-free holes to serve as blank control; after the end of the drug treatment, 10. Mu.l of CCK-8 solution was added to each well and the incubation was continued at 37℃for 2 hours, absorbance was measured at 450nm, and the experimental results were expressed as cell viability: cell viability (%) = (drug-treated group-blank)/(non-drug-treated group-blank) ×100%.
3. Conclusion of the experiment
FIG. 12A SGC7901 of CGA genes WT and KO ADR Cell growth curves of cells treated by fluorouracil and doxorubicin show that after CGA gene knockout, gastric cancer drug-resistant cell strain SGC7901 ADR The proliferation capacity of fluorouracil and doxorubicin under the treatment condition is obviously reduced; FIG. 12B SGC7901 of CGA genes WT and KO VCR Cell growth curve of cells treated by fluorouracil and taxol shows that after CGA gene knockout, gastric cancer drug-resistant cell strain SGC7901 VCR The proliferation capacity of fluorouracil and taxol under the treatment condition is obviously reduced; as can be seen from fig. 12, CGA gene knockout can result in a significant decrease in the proliferative capacity of gastric cancer resistant cells in the chemotherapeutic drug environment.
Example 13: knockout of CGA Gene on gastric cancer resistant cell line SGC7901 ADR And SGC7901 VCR Effects on chemotherapeutic drug treatment in mice
1. Study object
Construction of a successful gastric cancer resistant cell line SGC7901 Using example 9 ADR And SGC7901 VCR Monoclonal cell line (KO-1) knocked out CGA gene with multidrug resistant WT SGC7901 ADR And SGC7901 VCR Cells served as control; the experimental animals were selected from 6-8 week old female thymus-deficient nude mice (purchased from Experimental animal technologies Co., ltd., violet, beijing) and kept in a constant temperature and humidity SPF environment.
2. Experimental method
SGC7901 employing CGA gene WT or KO ADR And SGC7901 VCR Cells were subjected to a nude mouse subcutaneous oncology experiment and their effect on tumor growth (tumor volume and weight) was observed by intraperitoneal injection of chemotherapeutic drugs. The specific experimental method is as follows: nude mice were randomly divided into control groups (SGC 7901 injected with CGA gene WT ADR And SGC7901 VCR Cells) and experimental groups (SGC 7901 injected with CGA gene KO ADR And SGC7901 VCR Cells), 5 nude mice each, 5X 10 injections under the right flank of each nude mouse 6 A cell; after 2-3 weeks of tumor cell tumor injection, the tumor is grown to about 100mm 3 At this time, the control group and the experimental group were administered to the abdominal cavities of nude mice, respectively, every 3 days with physiological saline, fluorouracil (20 mg/kg), doxorubicin (8 mg/kg) or paclitaxel (3 mg/kg), and the size of the formed tumor was observed and measured (tumor volume=0.52×long diameter×short diameter) 2 ). On the day of termination of the experiment, mice were sacrificed and tumors were weighed as required by the theory of experimental animals.
IHC staining is carried out after tumor tissue is fixed, embedded and sectioned, and the expression of cell proliferation related protein Ki-67 and apoptosis related protein clean Caspase-3 is observed. The IHC comprises the following specific steps: placing the tissue slice in a 60 ℃ incubator, baking for about 1h, dewaxing in xylene for 10min, repeating for 3 times, and sequentially placing in 99.9%, 96% and 70% ethanol for 5min for dehydration, repeating for 3 times; boiling the slices in sodium citrate antigen retrieval solution for 15min, and placing the slices in 3%H 2 O 2 Dripping the sealing liquid for incubation for 30min at room temperature for 15 min; primary antibody (rabbit anti-human Ki-67 mab, abcam,1:200; rabbit anti-human clear Caspase-3 mab, abcam, 1:200) was incubated overnight at 4 ℃. Washing with PBS for 5min, repeating for 3 times, and incubating the secondary antibody (biotin-labeled goat anti-rabbit IgG polymer, meta fir gold bridge) for 1h at room temperature. PBS washing, DAB, developing, and staining cell nuclei with hematoxylin; after 1% hydrochloric acid alcohol is differentiated and 0.1% ammonia water returns to blue, the slices are sequentially placed in 70%, 96% and 99.9% ethanol for dehydration, and a neutral resin sealing piece is used.
3. Conclusion of the experiment
FIGS. 13A and 13B are SGC7901 of CGA genes WT and KO ADR And SGC7901 VCR Tumor growth curve of cells in nude mice under conditions of treatment with fluorouracil, doxorubicin and paclitaxel. FIGS. 13C and 13D SGC7901 of CGA genes WT and KO ADR And SGC7901 VCR Tumor pictures and tumor weight statistics of cells under therapeutic conditions for fluorouracil, doxorubicin and paclitaxel in nude mice. FIGS. 13A-D show that gastric cancer drug-resistant cell strain SGC7901 after CGA gene knockout ADR And SGC7901 VCR The growth in vivo against chemotherapeutic drugs is significantly slowed down. FIGS. 13E and 13F are SGC7901 of CGA genes WT and KO ADR And SGC7901 VCR Tumor cells treated by fluorouracil, doxorubicin and taxol in nude mice has representative microscopic images after being stained with Ki-67 and clear Caspase-3 protein IHC, which shows that after CGA gene knockout, gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR After in vivo treatment by chemotherapy drugs, the proliferation capacity is obviously weakened, and the apoptosis is obviously increased. As can be seen from fig. 13, CGA gene knockout can result in a significant decrease in the ability of gastric cancer resistant cells to resist chemotherapeutic drugs in vivo.
Example 14: interfering with expression of CGA gene by microRNA (miRNA) in gastric cancer cells
1. Study object
Gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR Is the same as in example 1; mirnas are non-coding small molecule RNAs of about 18-25 nucleotides in length within eukaryotic cells, often negatively regulating gene expression at posttranscriptional levels. Chemically synthesized miRNA mimics of the present invention were purchased from guangzhou sharp biotechnology limited.
2. Experimental method
miRNAs capable of specifically targeting CGA genes are respectively predicted through online miRNA prediction algorithm websites miRWalk (http:// miRWalk. Umm. Uni-heidelberg. De) and targetScan (http:// www.targetscan.org/vert_72 /), and the intersection of the two is taken to obtain 36 miRNAs of the specific targeting CGA genes (Table 8). The function of these 36 miRNAs in cancer resistance was verified one by Pubmed (https:// Pubmed. Ncbi. Lm. Nih. Gov), and 4 miRNAs (miR-17-3 p, miR-630, miR-708-3p, miR-761) whose function in gastric cancer resistance was not reported were selected for subsequent study.
RT-qPCR detection of 4 miRNAs on gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR The expression difference between the parent chemotherapy sensitive SGC7901 cells is as follows: after each cell was cultured for 24 hours, total RNA was extracted from three cells using QIAGEN RNA extraction kit, respectively, and the concentration and quality of RNA was measured using a spectrophotometer; reverse transcription of RNA into cDNA using Takara reverse transcription kit, reverse transcription system: RNA 3.75. Mu.l, 2 XmRQ Buffer 5. Mu.l, mRQ Enzyme 1.25. Mu.l; reverse transcription reaction conditions: incubation at 37℃for 60min,85℃for 5min, and 4℃for maintenance; using Takara SYBR Premix Ex Taq II with the reverse rate product as a template, performing RT-qPCR detection by a chimeric fluorescence method, and reacting the reaction system: 2X SYBR Premix Ex Taq II. Mu.l, 1. Mu.l upstream primer, 1. Mu.l downstream primer, 2. Mu.l cDNA, ddH 2 O was added to the reaction volume to 20. Mu.l; reaction conditions: reacting at 95 ℃ for 30s, reacting at 95 ℃ for 5s, reacting at 60 ℃ for 30s (45 cycles), and collecting fluorescence at 60 ℃; PCR reaction primers for each miRNA were purchased from the guangzhou sharp biotechnology company. By 2 -△△CT And calculating a quantitative result by a method, carrying out statistical analysis on the expression quantity of each mRNA in each cell strain, wherein CT is the number of cycles required for the fluorescent signal to reach a threshold value.
The mimic of 4 miRNAs is transfected into gastric cancer drug-resistant cell strain SGC7901 by adopting a liposome method ADR And SGC7901 VCR The method comprises the following specific steps: SGC7901 ADR And SGC7901 VCR Culturing the cells to logarithmic phase, digesting each cell with pancreatin, inoculating into 6-well plate, and counting cells per well by about 2×10 5 A plurality of; when the growth fusion of the cells in the 6-well plate reaches about 30%, the culture medium is replaced by Opti-MEM culture medium; opti-MEM (available from Gibco) was separated from m in corresponding proportions using a sterile microcentrifuge tubeMixing iR-17-3p, miR-630, miR-708-3p, miR-761 analogue and negative control uniformly; another microcentrifuge tube was taken and mixed well with Opti-MEM and Lipofectamine RNAiMAX (available from Invitrogen) in the corresponding proportions; after the two pipes are incubated for 5min at room temperature, the two pipes are uniformly mixed and incubated for 20min at room temperature; slowly adding the mixed solution into a corresponding 6-hole plate, and changing the mixed solution into a corresponding normal culture medium after 48 hours; SGC7901 detection Using WB ADR And SGC7901 VCR Expression of CGA protein in cells transfected with 4 miRNA mimetics was performed in the same manner as in example 2.
3. Experimental results
Fig. 14A is a schematic representation of a screening strategy for specific mirnas targeting CGA genes. FIG. 14B shows miR-17-3p, miR-630, miR-708-3p and miR-761 in gastric cancer drug-resistant cell SGC7901 ADR And SGC7901 VCR And the expression of the 4 miRNAs in parent SGC7901 cells thereof show that the expression of the 4 miRNAs in drug-resistant cells is reduced, which is a precondition for up-regulating the miRNAs to inhibit CGA in a targeted manner. FIG. 14C shows the effect of SGC7901 on gastric cancer resistant cells ADR And SGC7901 VCR After being transfected with the analogs of miR-17-3p, miR-630, miR-708-3p and miR-761, WB detects the expression change of the CGA protein in cells, which indicates that miR-708-3p and miR-761 can obviously inhibit the expression of the CGA protein and plays a role in targeted inhibition of the CGA. As can be seen from fig. 14, the miRNA can interfere with the expression of CGA gene in gastric cancer cells, wherein miR-708-3p and miR-761 are mirnas effective in inhibiting CGA.
TABLE 8 MiRWalk and TargetScan databases together predict CGA-targetable miRNAs
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Example 15:miRNA interference CGA gene on gastric cancer drug-resistant cell line SGC7901 ADR And SGC7901 VCR Influence of cell viability under different treatment with therapeutic drugs
1. Study object
Gastric cancer drug-resistant cell strain SGC7901 ADR And SGC7901 VCR Is the same as in example 1. Chemically synthesized analogs of miR-708-3p and miR-761 were purchased from Guangzhou Ruibo Biotechnology Co.
2. Experimental method
Transfection of miR-708-3p and miR-761 mimetic into gastric cancer drug resistant cell strain SGC7901 by adopting liposome method ADR And SGC7901 VCR The specific procedure is the same as in example 9; cell viability assay to transfect SGC7901 of negative control miRNA mimics with IC50 concentration treatment of fluorouracil, doxorubicin, paclitaxel ADR And SGC7901 VCR Cells were used as controls and the procedure was as in example 10.
3. Conclusion of the experiment
FIG. 15A is SGC7901 transfected with miR-708-3p and miR-761 mimetic ADR Cell viability under fluorouracil and doxorubicin treatment, indicating that transfected miR-708-3p and miR-761 mimetics interfere with CGA gene expression, gastric cancer drug resistant cell line SGC7901 ADR The viability of fluorouracil and doxorubicin treatment conditions was significantly reduced. FIG. 15B is SGC7901 transfected with miR-708-3p and miR-761 mimetic VCR Cell viability under fluorouracil and paclitaxel treatment, indicating that transfected miR-708-3p and miR-761 mimetics interfere with CGA gene expression, gastric cancer drug resistant cell line SGC7901 VCR The viability of fluorouracil and doxorubicin treatment conditions was significantly reduced. As can be seen from fig. 15, interference of CGA gene expression by miRNA can lead to a significant decrease in the viability of gastric cancer resistant cells in the chemotherapeutic drug environment.
Example 16: miRNA interference CGA gene on gastric cancer drug-resistant cell line SGC7901 ADR Effects on chemotherapeutic drug treatment in mice
1. Study object
Gastric cancer drug-resistant cell strain SGC7901 ADR Is the same as in example 1. Experimental movementThe sources and feeds were the same as in example 13. The miR-708-3p and miR-761 mimetics for in vivo injection are purchased from the open biosciences of Western An Rongqing.
2. Experimental method
Adopts gastric cancer drug-resistant cells SGC7901 ADR The subcutaneous nodulation experiment of nude mice is carried out, and the specific implementation steps are as follows: 5X 10 injections were given subcutaneously on the right flank of each nude mouse 6 Individual cells. After 2-3 weeks of tumor cell tumor injection, the tumor is grown to about 100mm 3 At the time, the nude mice are randomly divided into 6 groups of 8 nude mice each; group 1 intratumorally injected miRNA control drug (produgctrl) and intraperitoneal injection of normal saline every 3 days; group 2 intratumorally injected miRNA control drug and intraperitoneally fluorouracil (20 mg/kg) every 3 days; intratumorally injecting miR-708-3p mimic drug (miR-708-3 p prodrug) and intraperitoneal injection of normal saline every 3 days in group 3; intratumorally injecting miR-708-3p analogue medicament and intraperitoneal injecting fluorouracil every 3 days in group 4; intratumorally injecting miR-761 mimetic medicament (miR-761 pro drug) and intraperitoneal injection of normal saline every 3 days in group 5; intratumoral administration of miR-761 mimetic agents and intraperitoneal administration of fluorouracil every 3 days in group 6. The size of the formed tumors of each group was observed and measured (tumor volume=0.52×long diameter×short diameter 2 ). On the day of termination of the experiment, mice were sacrificed and tumors were weighed as required by the theory of experimental animals. IHC staining is carried out after tumor tissue is fixed, embedded and sectioned, and the expression of the cell proliferation related protein Ki-67 and the apoptosis related protein clean Caspase-3 is observed, and the specific steps are the same as in example 5.
3. Conclusion of the experiment
FIG. 16A is SGC7901 ADR Tumor growth curves after cell neoplasia under 6 different treatment conditions, FIG. 15B is SGC7901 ADR Tumor pictures under 6 different treatment conditions after cell neoplasia, FIG. 16C is SGC7901 ADR Figures 16A-C show that after miR-708-3p and miR-761 mimetics are injected to interfere with CGA gene expression, a gastric cancer drug-resistant cell line SGC7901 ADR The growth in vivo against chemotherapeutic drugs is significantly slowed down; FIG. 16D is SGC7901 ADR Ki-67 and clear Caspas under 6 different treatment conditions after cell neoplasiaRepresentative micrograph of e-3 protein IHC after staining shows that after miR-708-3p and miR-761 analogue are injected to interfere with CGA gene expression, gastric cancer drug-resistant cell strain SGC7901 ADR The proliferation capacity of the cells is obviously weakened after the cells are treated by the chemotherapeutic drugs in vivo, and the apoptosis is obviously increased. As can be seen from fig. 16, the interference of miRNA with CGA gene expression can lead to a significant decrease in the ability of gastric cancer resistant cells to resist chemotherapeutic drugs in vivo.
Example 17: interfering with CGA protein glycosylation and gastric cancer resistant cell line SGC7901 ADR Influence of cell viability under treatment with chemotherapeutic drugs
Glycosylation is an important post-translational modification process after protein synthesis, and interference with the protein glycosylation process can affect the stability of the protein, thereby interfering with its expression in cells. The aim of this example is to demonstrate that interfering with glycosylation modification of CGA proteins can reduce the resistance of cells to chemotherapeutic drugs.
1. Study object
This example used the construction of a successful gastric cancer resistant cell line SGC7901 from example 9 ADR KO-1 monoclonal cell strain with CGA gene knocked out is prepared through adding rCGA treated in different conditions or CGA plasmid transfected with glycosylation modification site mutation into culture medium, and detecting cell survival rate under the condition of chemotherapeutic medicine treatment.
2. Experimental method
The rCGA solution (available from St. Johnsony, beijing) was incubated with peptide N-glycosidase F (PNGase F, available from St. Johnsony, beijing) and reacted at 37℃for 4h to remove N-desman on the peptide chain of the rCGA protein.
Two N-glycosylation sites (aspartic acid at positions 52 and 78) on a wild-type (WT) human CGA expression plasmid (purchased from Shanghai Ji Kai company) were mutated to glutamic acid (mutated aspartic acid at position 52 is designated N52Q, mutated aspartic acid at position 78 is designated N78Q, and mutated aspartic acids at positions 52 and 78 are designated DM) separately or simultaneously by the QuickChangePCR method, and the QuickChangePCR was performed in the same manner as in example 9.
SGC7901 knocked out at CGA ADR Adding PNGase F or not to the cell culture mediumrCGA (20. Mu.g/ml) of (A); or SGC7901 obtained by transfecting human CGA expression plasmid of WT, N52Q, N Q or DM with CGA gene by liposome method ADR Cells were prepared in the same manner as in example 9; after the above treatment, the cells were subjected to viability assay at IC50 concentrations of fluorouracil and doxorubicin, following the procedure of example 10.
3. Conclusion of the experiment
FIG. 17A is SGC7901 with CGA gene knocked out ADR Cell viability of cells after addition of rCGA under fluorouracil and doxorubicin treatment indicated SGC7901 with CGA gene knocked out after addition of rCGA without PNGase F treatment ADR The survival ability of fluorouracil or doxorubicin under the treatment condition is obviously enhanced; however, after PNGase F-treated rCGA was added, CGA gene knocked out SGC7901 ADR There was no significant difference in viability under fluorouracil or doxorubicin treatment conditions. FIG. 17B is SGC7901 with CGA knockdown ADR Cell viability under fluorouracil and doxorubicin treatment following transfection of cells with CGA expression plasmids for WT, N52Q, N78Q or DM, indicating CGA knock-out SGC7901 following transfection of CGA expression plasmid for WT ADR The survival ability of fluorouracil or doxorubicin is obviously enhanced under the treatment condition, but after the CGA expression plasmid of N52Q, N78Q or DM is transfected, the CGA gene knocked out SGC7901 ADR There was no significant difference in viability under fluorouracil or doxorubicin treatment conditions. As can be seen from fig. 17, interfering with CGA protein glycosylation can result in a significant decrease in the viability of gastric cancer resistant cells in the context of chemotherapeutic drugs, and can be used as a means of reducing tumor cell resistance.
Claims (9)
1. The application of glycoprotein hormone alpha polypeptide gene or the coded protein thereof as a target spot in preparing a reagent or a kit for diagnosing and/or prognosis evaluation of solid tumor drug resistance; the solid tumor is gastric cancer; the drug resistance is drug resistance to single chemotherapeutic drugs or multiple chemotherapeutic drugs.
2. The use according to claim 1, wherein the reagent or kit detects messenger RNA transcribed from glycoprotein hormone alpha polypeptide genes or glycoprotein hormone alpha polypeptide gene-encoded proteins 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 assay.
3. The use according to claim 1, wherein the reagent or kit comprises a specific primer for amplifying the glycoprotein hormone alpha polypeptide gene, a probe that hybridizes to the nucleotide sequence of the glycoprotein hormone alpha polypeptide gene, or an antibody or antibody fragment that specifically binds to the glycoprotein hormone alpha polypeptide.
4. The use according to claim 3, wherein the antibody is a monoclonal or polyclonal antibody.
5. The use according to any one of claims 1 to 4, wherein the test sample of the reagent or kit is serum, plasma, cells, cell culture supernatant, urine, tissue or tissue lysate.
6. Application of glycoprotein hormone alpha polypeptide gene expression inhibitor in preparing medicine for treating drug-resistant solid tumor; the glycoprotein hormone alpha polypeptide gene expression inhibitor is selected from CRISPR gene editing therapeutic drugs or antisense nucleic acid drugs which block normal transcription or posttranscriptional translation processes of glycoprotein hormone alpha polypeptide genes through CRISPR/Cas9 gene editing technology or RNA interference technology; the solid tumor is gastric cancer; the drug resistance is drug resistance to single chemotherapeutic drugs or multiple chemotherapeutic drugs.
7. The construction method of the drug-resistant solid tumor cell research model is characterized by comprising the following steps: knocking out glycoprotein hormone alpha polypeptide genes in solid tumor cells; culturing solid tumor cells in a culture medium containing one or more chemotherapeutic drugs to construct initial drug-resistant solid tumor cells, and knocking out glycoprotein hormone alpha polypeptide genes in the initial drug-resistant solid tumor cells to obtain drug-resistant solid tumor cells; the solid tumor is gastric cancer.
8. The method for constructing a research model of drug-resistant solid tumor cells according to claim 7, wherein the glycoprotein hormone alpha polypeptide gene in the initial drug-resistant solid tumor cells is knocked out by using CRISPR/Cas9 technology to obtain the drug-resistant solid tumor cells.
9. The construction method of the drug-resistant solid tumor animal model is characterized by comprising the following steps: injecting the drug-resistant solid tumor cells constructed by the method of claim 7 or 8 into an animal model to construct a drug-resistant solid tumor animal model.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2667364A1 (en) * | 2007-01-16 | 2008-07-24 | Musc Foundation For Research Development | Compositions and methods for diagnosing, treating, and preventing prostate conditions |
| CN101868534A (en) * | 2007-08-10 | 2010-10-20 | 哈布勒支研究所 | Be used to identify, increase and remove the method for adult stem cell and cancer stem cell |
| CN104822844A (en) * | 2012-10-01 | 2015-08-05 | 米伦纽姆医药公司 | Biomarkers and methods to predict response to inhibitors and uses thereof |
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| US20060234246A1 (en) * | 1999-02-02 | 2006-10-19 | Chiron Corporation | Gene products differentially expressed in cancerous cells |
| EP1167975B1 (en) * | 2000-06-22 | 2009-10-07 | Universite Paris Descartes | Compositions and methods for detecting, treating or predicting the response of breast tumor cells to endocrine therapy |
| US20070092881A1 (en) * | 2003-07-10 | 2007-04-26 | Central Institute For Experimental Animals | Gene markers of tumor metastasis |
| AU2004287645A1 (en) * | 2003-11-06 | 2005-05-19 | Compugen Ltd. | Variants of human glycoprotein hormone alpha chain: compositions and uses thereof |
| US20110016541A1 (en) * | 2008-12-04 | 2011-01-20 | Sigma-Aldrich Co. | Genome editing of sensory-related genes in animals |
| CA2791905A1 (en) * | 2010-03-01 | 2011-09-09 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Biomarkers for theranostics |
| CN105671147A (en) * | 2015-12-31 | 2016-06-15 | 中国医科大学附属第一医院 | TRbeta gene mutation detection kit and TRbeta gene mutation detection method |
| US20180126003A1 (en) * | 2016-05-04 | 2018-05-10 | Curevac Ag | New targets for rna therapeutics |
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| CN101868534A (en) * | 2007-08-10 | 2010-10-20 | 哈布勒支研究所 | Be used to identify, increase and remove the method for adult stem cell and cancer stem cell |
| CN104822844A (en) * | 2012-10-01 | 2015-08-05 | 米伦纽姆医药公司 | Biomarkers and methods to predict response to inhibitors and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| Chromogranin A regulates neuroblastoma proliferation and phenotype;Dongyun Zhang et al;《Biology Open》，;第第8卷卷;第1-12页 * |
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