| Objective :1.To analyze the expression of EZH2 in gastric cancer and its relationship with clinicopathological parameters and chemotherapy efficacy through public database,gastric cancer tissues and gastric cancer cells.2.The effects of EZH2 gene on the growth and sensitivity to chemotherapy of gastric cancer were studied in vivo and in vitro.3.To explore the related molecular biological mechanisms and signaling pathways of EZH2-mediated growth and drug resistance of gastric cancer.Methods:1.The expression of EZH2 gene in gastric cancer tissues was analyzed by bioinformatics method in public database.Quantitative reverse transcription polymerase chain reaction(RT-PCR)and Western blot were used to detect the expression of EZH2 m RNA and protein in gastric cancer cells.The expression of EZH2 protein in gastric cancer tissues was detected by immunohistochemistry,and its correlation with clinicopathological parameters,chemotherapy response rate and progression-free survival was analyzed.2.Lentiviral vector sh EZH2 was constructed,and then human gastric cancer cell line MGC803 with high expression of EZH2 was infected by lentivirus,and the stable transgenic strain was screened out.Real-time Q-PCR and Western blot were used to detect the interference effect.3.In vitro cytological experiments: CCK-8 proliferation test,Transwell compartment invasion test and scratch test were used to test the changes of proliferation,invasion and migration of human gastric cancer cell line MGC803 before and after EZH2 silencing.CCK-8 toxicity test was used to compare the sensitivity of EZH2 to paclitaxel,oxaliplatin and 5-fluorouracil before and after EZH2 silication,and the synergism of EZH2 inhibitor GSK126 with chemotherapy drugs.In vivo study,EZH2-silenced human gastric cancer cell line transplanted tumor model was established in nude mice,and the effects of EZH2 down-regulation on gastric cancer growth and sensitivity to chemotherapy were studied by intrapitoneal infusion of paclitaxel.4.First,The Cancer Genome Atlas(TCGA)transcriptome dataset was used for KEGG signaling enrichment analysis to explore downstream pathways regulated by EZH2 activation.Secondly,the molecular mechanisms and signaling pathways of EZH2 in gastric cancer were further explored by cell transcriptome sequencing analysis.Then Western blot assay was used to verify the effect of EZH2 down-regulation on the expression of key proteins in the signaling pathway.Finally,the effects of down-regulation of EZH2 on the downstream growth and drug-resistance related molecules of the signaling pathway were detected by real-time Q-PCR technology,so as to explore the molecular mechanism related to the growth and drug resistance of gastric cancer.Results:1.Results from public databases showed that EZH2 expression in gastric cancer was higher than that in normal tissues or tissues adjacent to the corresponding cancer.EZH2 m RNA and protein of MGC803,BGC823,AGS and HGC27 gastric cancer cell lines were all expressed,and MGC803 had the highest expression.Immunohistochemical results showed that EZH2 gene was highly expressed in advanced gastric cancer,and EZH2 protein expression was correlated with pathological types and chemotherapy efficacy,and EZH2 protein expression was negatively correlated with chemotherapy efficacy.The median TTP of the group with high EZH2 expression was lower than that of the group with low EZH2 expression,with statistical difference.2.EZH2-sh RNA lentiviral vector was successfully constructed and transfected into human gastric cancer cell line MGC803.Real-time Q-PCR and Western blot detection of interference effect proved the successful construction of gastric cancer cell line MGC803 with stable silencing of EZH2.3.In vitro cytology experiment: The results of CCK-8 proliferation experiment showed that the OD value and cell proliferation rate in the EZH2-silenced group were significantly lower than those in the control group,and the difference was statistically significant.Transwell assay showed that compared with the control group,the number of metastatic cells was significantly reduced in the EZH2-silenced group,which was statistically significant.The scratch damage experiment showed that,compared with the control group MGC803-Vector,the scratch healing rate of MGC803-sh EZH2 cells decreased from 12 h to 36 h,and the differences were statistically significant.CCK8 toxicity assay showed that EZH2 silences increased the sensitivity of MGC803 cells to paclitaxel,oxaliplatin and5-fluorouracil,and the IC50 values were significantly decreased,with statistically significant differences.It can be seen that the inhibition effect of paclitaxel,oxaliplatin and 5-fluorouracil on MGC803-sh EZH2 cells was enhanced after EZH2 gene silencing.The synergistic study showed that EZH2 inhibitor GSK126 had synergistic effects with paclitaxel,oxaliplatin and 5-fluorouracil.Among them,the CI value of GSK126 combined with paclitaxel increased with the increase of paclitaxel dose,indicating that the small dose of paclitaxel and GSK126 showed better synergistic effect.In vivo tumor transplantation experiments in nude mice,it was found that silenced EZH2 reduced the subcutaneous tumor-forming ability of gastric cancer cell line MGC803 in nude mice and increased the sensitivity to paclitaxel chemotherapy.4.KEGG signal pathway enrichment analysis in public database showed that EZH2 may mediate the biological characteristics of gastric cancer through m TOR signal transduction,MAPK signal transduction,P53 signal transduction and erb B signal transduction.Further transcriptome sequencing analysis showed that 1735 DEGs were screened and identified before and after EZH2 silencing,of which 1131 were up-regulated and 604 were down-regulated.GO functional enrichment analysis showed that up-regulated DEGs were mainly related to targeted membrane protein translation,cytoplasmic translation,ribonucleic acid metabolism,mitochondrial electron transport,etc.,while down-regulated DEGs were mainly related to the regulation of growth and development,signaling pathway,phosphorylation,and protein kinase C activity.KEGG signaling pathway enrichment analysis showed that there were 19 significant signaling pathways,including tumor pathway,PI3K-Akt signaling pathway,FOXO signaling pathway,erb B signaling pathway,HIF-1signaling pathway,TGF-β signaling pathway and so on.Furthermore,Western blot assay confirmed that EZH2 silencing down-regulated the expression of PI3 K and p-Akt protein in MGC803 gastric cancer cells,while the total expression of Akt did not significantly change,suggesting that EZH2 down-regulation can block the phosphorylation process of Akt,thereby inhibiting the over-activation of p13k-Akt signaling pathway.Real-time Q-PCR was used to detect the molecular mechanism of EZH2 in regulating gastric cancer growth and drug resistance.The results showed that EZH2 silencing up-regulated the expression of apoptotic molecule p53 m RNA and down-regulated the expression of Bcl-2 m RNA in gastric cancer.MDR-1,MRP-1,LRP,BCRP and GST-π m RNA expression were down-regulated in gastric cancer,and the differences were statistically significant.Conclusion:1.EZH2 is highly expressed in gastric cancer,and the high expression of EZH2 may be related to tumor progression,poor prognosis and poor sensitivity to chemotherapy.2.Human gastric cancer cell line MGC803 with stable silencing of EZH2 was successfully constructed,laying a foundation for subsequent experiments.3.In vivo and in vitro experiments have confirmed that EZH2 silencing can slow the growth of gastric cancer,reduce the invasion and migration ability,and improve the sensitivity to chemotherapy drugs.4.EZH2 inhibitor GSK126 has synergistic effects with paclitaxel,oxaliplatin and5-fluorouracil,and EZH2 may become a new therapeutic target for gastric cancer.5.The activation of PI3K-Akt signaling pathway by EZH2 is one of the important mechanisms of gastric cancer;EZH2 mediates the growth and drug resistance of gastric cancer by regulating the expression of apoptosis-related molecules and drug-resistant molecules. |
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