The Expression Level And Biological Function Of N6-Methyladenosine Methyltransferase-Like 3 In Human Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world.The annual incidence of HCC ranks fifth and the mortality ranks second.Due to the high invasive rate,frequent intrahepatic spread,and inadequacy for standard radiotherapy or chemotherapy,the five-year survival rate of patients with advanced HCC is less than 10%.Therefore,exploring the molecular mechanism of HCC development proves to be quite essential to take effective preventive or therapeutic measures,and then reducing the morbidity and mortality.Epigenetic variation plays an important role in the development of human cancer,including DNA methylation,histone modification,chromatin remodeling and RNA silencing.In recent years,diversified and reversible RNA modifications has been confirmed as a new field of epigenetic regulation.N6 methyladenylatin modification(m~6A),a kind of methylation modifications occurring at the sixth N atom of adenine,is the most common and abundant chemical modification in eukaryotic RNA.It is mainly consist of methyltransferase complex,demethylase and the corresponding reader,which in general maintains the stability of the RNA and also regulates splicing and translation.It proves to be active in many pathophysiological processes,such as tumors,reproduction,biological rhythms and so on.Methyltransferase 3(METTL3)as the core component,together with METTL14 and WTAP,constitutes a m6A methyltransferase complex,which mediates the m6A modification of RNA and LncRNA.Several studies have confirmed that METTL3 is heterogeneously expressed in leukemia,malignant glioma,lung cancer,liver cancer and breast cancer,and is highly associated with prognosis of patients.In addition,its role in different tumors is not entirely consistent.At present,whether METTL3-mediated m6A modification is differentially expressed in HCC,its biological function and specific molecular mechanism are still unclear.In this study,differential expression,biological function as well as possible regulation mechanism of METTL3 in HCC were studied.This study aimed at offering preliminary experimental data for further in-depth study of the specific molecular pathways of m6A in HCC occurrence and development,hoping to present new viewpoints for future targeted treatment.Methods1 METTL3 expression and its clinical significance in HCC1.1 TCGA database was used to analyze the expression of METTL3 in HCC and normal liver tissues,as well as in different clinical stages and pathological grades of hepatocellular carcinoma,and to analyze the relationship between METTL3expression level and the prognosis of patients.1.2 Immunohistochemical detection of HCC microarry was analyzed to verify the consistency with TCGA database analysis.2 The effect of METTL3 on the biological function of HCC cells2.1 Two HCC cell lines,namely HepG2 and Huh-7 were selected.Small interfering RNA(si-RNA)was used to down-regulate METTL3 expression by liposome transfection(si-METTL3 group).The transfection efficiency was detected by qRT-PCR and Western-Blot.Non-transfected cell lines were regarded as normal control group(siNC group).2.2 Cell cloning and CK-8 experiments were used to detect the proliferation ability of HCC cells in each group,while Cell scratch and Transwell experiments were used to detect the migration ability.3 Possible downstream regulation mechanism of METTL3TCGA database was used to find genes co-expressed with METTL3 in HCC,and then GO enrichment analysis was carried out.Combined with previous literature,possible pathways of METTL3 were speculated.Results1 METTL3 expression and its clinical significance in HCC1.1 The results of TCGA database showed that the expression of METTL3 in HCC was significantly higher than that in normal liver tissues;the expression of METTL3in stage III was significantly higher than that in stage I and II,and the expression of METTL3 in grade III was significantly higher than that in grade I and II;the overall survival time of patients with high expression of METTL3 was significantly lower than that of patients with low expression,all P<0.05.1.2 The IHC results were basically consistent with the above results.2 Effect of METTL3 on biological function of HCC cells2.1 The expression levels of METTL3 mRNA and protein in siMETTL3-1 and siMETTL3-2 groups were significantly decreased.2.2 Compared with the siNC group,the proliferation and migration ability of cells in siMETTL3 group was significantly enhanced according to cell cloning and CCK-8assay,Cell scratch and Transwell experiments.3 Possible downstream regulation mechanism of METTL3METTL3 was positively correlated with RBBP4 expression,and RBBP4 inhibited HCC proliferation,invasion and metastasis through PTEN/AKT pathway.Conclusion1)METTL3 was highly expressed in human hepatocellular carcinoma.2)METTL3 level was correlated with clinical stage,pathological grade and prognosis of hepatocellular carcinoma.3)METTL3 promotes the proliferation and migration of human HCC cells.4)METTL3 may play these roles through METTL3-RBBP4-PTEN/AKT pathway.