Molecular Mechanism Of Lentiviral-mediated EGFR Expression Interference On Proliferation Of Gastric Cancer AGS Cells

ObjectiveFor the past few years,the outbreak rate of malignancy has increased year by yaar,especially the rate of gastric cnacer.Because of the low security of foodstuff,breakout rate of gastric cancer keeps all-time high.At the same time,the death rate of gastric caner has reaches the top three.It has fatal hazard to our human health.Most gastric cancers are diagnosed in the advanced stage,and chemotherapy has become an important treatment.However,standard chemotherapy for advanced gastric cancer has not yet been established.Targeted therapy has had little effect in recent years,and new methods and methods are urgently needed.Advances in molecular oncology bring hope to gastric cancer typing and individualized precision treatment,and gene interference technology has important therapeutic application prospects.EGFR is closely related to the development of gastric cancer.Previous studies in our group found that EGFR expression interference can affect the biological behavior of gastric cancer cells and the sensitivity of chemotherapy drugs.However,the molecular mechanism is still unclear.The aim of this study was to construct an EGFR-interfering gastric cancer AGS cell stable strain by lentivirus-mediated CCK-8 method to observe the effect of EGFR interference on the sensitivity of paclitaxel,oxaliplatin,fluorouracil,cisplatin and doxorubicin.Western-blot method was used to detect the expression changes of Akt,Bax,Bcl-2,BRCA1,Stathmin-1,Thymidylate Synthase and c-Myc in cells before and after EGFR interference,and the effect of EGFR interference on mRNA expression profile of gastric cancer AGS cells was detected by lncRNA microarray.Screening differentially expressed genes,analyzing the possible molecular mechanisms,and providing experimental and molecular theoretical basis for exploring the treatment of EGFR interference in the treatment of gastric cancer.MethodAGS cells were used as the research object to construct stable gastric cancer cell lines with low expression of EGFR by EGFR-siRNA interference method;the interference effect was verified by QPCR method;paclitaxel,oxaliplatin,fluorouracil and cisplatin were observed by CCK-8 method.And the effects of drug inhibition inhibition such as doxorubicin;the relative expression of Akt,Bax,Bcl-2,BRCA1,Stathmin-1,Thymidylate Synthase,and c-Myc were detected by Western-blot method.And use lncRNA chip to detect changes in related mRNA expression.Results1.After EGFR-siRNA was transfected into normal AGS cells,it was shown by QPCR that EGFR had interference effects in AGS cells,and EGFR-sh interference was down-regulated by 66%.2.Different concentrations of paclitaxel,oxaliplatin,fluorouracil,cisplatin and doxorubicin were used to treat the two groups of cells(AGS cell group and EGFR-siRNA AGS cell group)for 48 hours.The results showed that the inhibition rates of proliferation increased with the increasing of concentrations,and the inhibition rate of each concentration of EGFR-siRNA AGS cells was higher than that of AGS group(P<0.05).3.Western-blot results showed that the expression of AKT,Bcl-2,Stathin-1,Thymidylate Synthase and c-Myc in EGFR-siRNA AGS cells was lower than that in AGS cells,while the expression of Bax and BRCA1 was higher than that in AGS cells.Statistical significance(P<0.05)4.The lncRNA chip test results show that compared with AGS cells,242 genes were up-regulated in EGFR-siRNA AGS cells,and 641 genes were down-regulated.The top 10 genes whose expression is up-regulated are IFI27,FAM129 C,TRABD2B,WT1,XRCC4,NECTIN4,JCAD,CARD11,ACTA2 and GSDME.The top 10 genes whose expression is down-regulated are PLXNA2,RALGPS1,SHROOM2,HKDC1,GLI2,CEACAM6,MYB,SIRPB1,KCNE3 and NCF2.Pathway analysis revealed that the biological effects of EGFR expression inhibition may be related to the NF-κB signaling pathway.Conclusion1.EGFR gene interference can significantly increase the inhibitory effect of common chemotherapeutic drugs on the proliferation inhibition of gastric cancer AGS cells,which may be achieved by affecting the expression of cell proliferation regulatory genes and drug sensitivity-related genes.2.EGFR gene interference can lead to changes in the expression profile of gastric cancer AGS cells,and its biological effects may be related to signaling pathways such as NF-κB.