Molecular Mechanism Of Autophagy By Methyltransferase Regulatory Subunit Wtap In Hepatocarcinoma Cells

Liver cancer is a frequently occurring malignancy,and it ranks among the highest in the world in terms of cancer-related death.Our country is characterized by a high incidence of liver cancer.The incidence and number of liver cancer in our country ranks first in the world,and the situation of liver cancer prevention and treatment is still very serious.Autophagy is an evolutionarily conserved intracellular degradation and metabolism process.It plays a dual role in tumor cells.On the one hand,autophagy can induce autophagic death of tumor cells and inhibit the growth of tumor cells.On the other hand,autophagy can also protect tumors from chemotherapeutic drugs and delay tumor cell apoptosis.Therefore,it is a good strategy to improve the efficacy of cancer therapy through autophagy-induced tumor cell death.Meanwhile,it can provide important theoretical significance and medical value in tumor clinical treatment.N6-methyladenosine(m~6A)is a very conservative RNA modification in mammals.This modification is a dynamic and reversible process and has a high abundance in mammalian cells and m~6A plays an important role in all stages of the RNA life cycle.And m~6A is closely related to tumor occurrence and development.N6-methyladenosine(m~6A)has been reported to be capital of regulating autophagy,which can affect tumor growth and development.However,there is no relevant report on the regulation of the methyltransferase complex component WTAP on autophagy,and the relationship between the occurrence and development of liver tumor and WTAP remains unclear.To evaluate the effect of WTAP on autophagy,two hepatocellular carcinoma cell(HCC)lines BEL-7404 and SMMC-7721 with high WTAP expression were selected in the current study.Western blot and fluorescence observation showed that knockdown of WTAP could promote the occurrence of autophagy in HCC.To identify the proteins regulated by m~6A to modulate the autophagy level,two groups of total RNAs extracted from sh CON and sh WTAP cells were selected for high-throughput sequencing.Bioinformatics analysis showed that differential expression of some genes mainly existed in the AMPK signaling.As we know,the AMPK pathway is a well-known pathway.In the study,the AMPK pathway was identified to be involved in regulation of autophagy when knockdown of WTAP was made in liver cancer cells.Western blot was performed to examine the expression levels of some autophagy-related genes when the knockdown of WTAP was made in HCC.The results showed that the expression of p-AMPK protein was increased significantly compared with that of control group.Furthermore,the inhibitor Compound C was used to inhibit the phosphorylation of AMPK in cells.Western blot results confirmed that the agent could decrease the expression of p-AMPK in sh WTAP-treated cells.Fluorescence observation showed that Compound C could decrease the level of autophagy in sh WTAP-treated cells.As we know,LKB1 is an upstream kinase of AMPK.To further study the mechanism by which m~6A affects the abundance of p-AMPK,it is a highlight to study the abundance of LKB1 transcript and its protein product in the study.Methylated RNA immunoprecipitation(Me RIP)results showed that the knockdown of WTAP could reduce intracellular m~6A level.Furthermore,q PCR result showed that the knockdown of WTAP increased the abundance of LKB1 transcripts.Western blot confirmed that the knockdown of WTAP could facilitated the phosphorylation of AMPK by enhancing the stability of LKB1.Moreover,interference of LKB1(si LKB1)was made in sh WTAP-treated cells.The result showed that the expression level of p-AMPK was decreased in HCC through downregulation of LKB1.Additionally,sh WTAP could facilitate the autophagy in HCC but inhibite the proliferation of HCC.As we know,3-MA is an inhibitor that can efficiently block the formation of autophagosome.To further study the correlation between autophagy and cell proliferation,3-MA was used to treat the cells for analysis of cell proliferation.The results indicated that the number of sh WTAP-treated cells was significantly increased after treatment with 3-MA compared with that of sh WTAP group.Additionally,overexpression of WTAP in HCC was made to evaluate the ability of cell proliferation.Cell colony formation analysis showed that overexpression of WTAP promoted the proliferation of tumor cells.In conclusion,our results showed that the WTAP/LKB1/AMPK axis in HCC cells acts as a key regulator,linking m6 A with autophagy,which is directly involved with the proliferation of tumor cells.