The Role Of METTL3 In Promoting The Progression Of Hepatocellular Carcinoma Through Mediating ADH1A M6A Methylation

Objective: Analysis of DNA methylation and RNA m6 A methylesterase METTL3 mediating ADH1 A expression to promote malignant biological behavior in hepatocellular carcinoma(HCC)Methods:(1)Using TIMER and GENT2 databases with CPTAC and HPA sample databases to predict ADH1 A m RNA and protein expression levels in HCC and correlation of expression with clinical progression in HCC patients;using DNA methylation pan-cancer sequencing data from the SMART database to predict the degree of differential DNA methylation and expression correlation of ADH1 A.(2)To detect the methylation status and methylation level of ADH1 A in the tissues of HCC samples using Mass ARRY assay,to explore the differential Cp G sites regulating ADH1 A expression,and to analyze the correlation between ADH1 A methylation and clinical progression and prognosis of HCC in combination with clinicopathological parameters.(3)Western Blot and q RT-PCR were performed to analyze the correlation between METTL3/YTHDF2 and ADH1 A expression based on HCC m6 A prognostic model screening.(4)WB experiments were performed to detect changes in ADH1 A protein levels in cells after incubation with m6 A inhibitor(3-Deazaadenosine,3-DZA)and to infer the correlation between m6 A modifications and ADH1 A expression.(5)The ability of METTL3 to regulate ADH1 A protein expression was verified in WB assays after knockdown of METTL3.(6)CCK8 cell proliferation assay,Transwell invasion assay and cell scratch healing assay were performed in HCC cells to evaluate the ability of METTL3 to regulate HCC proliferation,invasion and migration.Result:(1)Bioinformatics data analysis showed that low/lost expression of ADH1 A m RNA and protein levels were significantly and positively correlated with the prognosis of HCC patients and clinical stage grading(P <0.001 for both),predicting that ADH1 A promoter region hypomethylation could upregulate ADH1 A expression in HCC(Cor <-0.2,P <0.05).(2)Mass ARRAY mass spectrometry analysis conducted in the subject HCC samples showed methylation modifications at the Cp G1-4 site in the ADH1 A promoter region,and the degree of Cp G2 methylation at the site was elevated in patients with low ADH1 A expression(P =0.037),but did not yet correlate with AD1 HA expression,patient prognosis and tumor stage grading(P >0.1).(3)In vitro cellular assays showed that METTL3 and YTHDF2 negatively regulated ADH1 A in HCC cells(Cor <-0.5,P <0.05).(4)After incubation of HCC cells with 3-DZA for 48 h,ADH1A protein expression level showed a dependent up-regulation with increasing 3-DZA concentration,and METTL3 and YTHDF2 expression were gradually down-regulated(P <0.05).(5)ADH1A protein expression level was significantly increased in MHCC97 H and PLC of hepatoma cells transfected with METTL3 small interfering RNA fragment(P <0.001).(6)The proliferation,migration and invasion abilities of HCC cells MHCC97 H and PLC were significantly increased after downregulation of METTL3 by small interfering RNA(P <0.05).Conclusion: METTL3 mediates the malignant biological behavior of HCC by regulating m6 A content in HCC cells to negatively regulate ADH1 A expression.