Sorafenib Induces Autophagy And Apoptosis Of Human Gastric Cancer Cell Line AGS By Regulating WTAP/PRDM1 Pathway

Objective: The clinical therapeutic effect of sorafenib in gastrointestinal tumors has been widely confirmed,but its mechanism in gastric cancer is still unclear.WTAP is the regulatory subunit of m6A methyltransferase,which can promote the occurrence and development of various malignant tumors,and it is involved in the regulation of the mechanism of action of a variety of cancer-targeted chemotherapy drugs.The purpose of this study was to explore the role of WTAP in the inhibition of human gastric cancer cell line AGS by the multi-molecular targeted drug sorafenib(SOR),and to further explore the possible mechanism.Method: The cytotoxicity of different concentrations of SOR on AGS cells and overexpression WTAP cells(OEWTAP)was determined by CCK-8 experiment.RNA-seq was used to determine the differential gene expression and enrichment pathway in group AGS,group AGS + SOR,group AGS＿OEWTAP and group AGS＿OEWTAP + SOR.The proliferative capacity of cells in each group was determined by plate cloning.The migration and invasion ability of cells in each group was measured by cell scratch test and Transwell migration and invasion test.The m RNA content of si PRDM1-1,si PRDM1-2,si PRDM1-3 in AGS group and WTAP in AGS group and AGS+SOR group were determined by qRT-PCR.ROS assay was used to detect the level of intracellular reactive oxygen species in AGS group and AGS+SOR group.JC-1 experiment detected the mitochondrial membrane potential level of OEWTAP group,OEWTAP+SOR group,OEWTAP knockdown PRDM1 group,and OEWTAP knockdown PRDM1 dosing group.WB assay was used to detect the changes of protein expression levels of WTAP,PRDM1,autophagy-related gene MAP1LC3 B,ATG16L1,ULK1,SQSTM1,apoptosisrelated gene TNFAIP1 and DNA repair enzyme PARP1 in different groups.Results: MTT results showed that compared with group AGS,the inhibition ability of SOR on group AGS＿OEWTAP was relatively weakened,with IC50 of 9.478 umol/L and 13.71 umol/L respectively.Plate cloning,scratch test and Transwell test showed that compared with group AGS,the proliferation,migration and invasion ability of group AGS＿OEWTAP was significantly increased;Compared with group AGS＿OEWTAP +SOR,the proliferation and migration of cells in group AGS + SOR were lower.The results of transcriptome sequencing analysis showed that the m RNA levels of WTAP,PRDM1,GASK1 A,H2AC11,SEMA6 B decreased after the addition of SOR in AGS cells,increased after the overexpression of WTAP,and decreased again after the overexpression of WTAP;At the same time,enrichment analysis showed that compared with AGS group,AGS+SOR group was positively correlated with DNA damage repair and autophagy(P<0.01).The protein level of WTAP did not change significantly after knocking down PRDM1,indicating that WTAP was regulating PRDM1.Western blot test found that there were differences in the expression of autophagy apoptosis-related genes MAP1LC3 B,ATG16L1,ULK1,SQSTM1,TNFAIP1,and PARP1 among group AGS,group AGS+SOR,group AGS＿OEWTAP,group AGS＿OEWTAP+SOR,group AGS＿si PRDM1,and group AGS＿OEWTAP＿si PRDM1+SOR.Drug treatment and knockdown of PRDM1 could increase the expression of autophagy apoptosis-related genes MAP1LC3 B,ATG16L1,ULK1,SQSTM1,and TNFAIP1,and decrease the expression of DNA repair enzyme PARP1,and drug treatment and knockdown of PRDM1 could overlap the protein expression changes.Conclusions: Overexpression of WTAP can promote the proliferation and migration of gastric cancer AGS cells.Sorafinil can inhibit PRDM1 by reducing the expression of WTAP,and then induce DNA damage repair,autophagy and apoptosis.