DNMT2 Knockdown Inhibits Hepatocellular Carcinoma By TNFSF10 Inducing Apoptosis

DNA methyltransferase 2(DNMT2)is a member of RNA methyltransferase,which catalyzes tRNA methylation and is closely related to development,epigenetic regulation,cellular metabolism.Recent studies have found that DNMT2 knockout can affect apoptosis of some cancer cells.Apoptosis is the active cell death controlled by the specific genes in order to maintain the stability of internal environment.Studies have shown that apoptosis is regulated by the endogenous(mitochondrial),exogenous(death receptor)and endoplasmic reticulum pathways mediated by apoptosis-related factors.Among them,apoptosis induced by tumor necrosis factor(TNFSF10)plays a key role in hepatocellular carcinoma,but its underlying mechanism remains unclear and needs further study.In this study,DNMT2 knockout HCC cells SMMC-7721 and Hepal-6 were constructed using CRISPR/Cas9 technology,and it was found that DNMT2 deletion inhibited the proliferation and migration of HCC cells.Our further stduies found that the early apoptosis was significantly enhanced,the mRNA and protein expression levels of apoptotic factor TNFSF10 were significantly up-regulated,and the expression levels of key proteases Caspase3,Caspase8,BAX and BCL-2 were changed in DNMT2-deficient cells.These results indicated that DNMT2 deletion activated apoptosis-related signaling pathways.In a mouse tumor model,we found that DNMT2 deletion inhibited hepatocellular carcinoma tumor growth and promoted apoptosis.In addition,Kaplan-Meier was used to analyze the correlation between DNMT2 expression and survival rate of HCC,which showed that the survival rate of HCC patients had a negative correlation with the level of DNMT2 expression.We further performed RNA-Seq high-throughput analysis,and found a potential target of DNMT2 in regulating apoptosis which was tumor necrosis factor TNFSF10.By constructing TNFSF10-RNAi stabilized cell lines,it was found that the level of apoptosis and cell phenotype were restored,suggesting that DNMT2 deletion promoted apoptosis by up-regulating TNFSF10 leading to inhibition of the HCC cell proliferation and migration.These results suggest a novel mechanism of DNMT2 in regulating hepatocellular carcinoma,and provide a new idea for further understanding the role of DNMT2 in hepatocellular carcinoma.