Preliminary Study On The Mechanism Of LncRNA MAPKAPK5-AS1 Inhibiting ADH1A By Binding EZH2 To Promote The Development Of Hepatocellular Carcinoma

Objective: To investigate the long noncodingRNA MAPKAPK5-AS1(lncRNA MAPKAPK5-AS1)recruitment gene enhancer homolog 2(EZH2)mediated alcohol dehydrogenase 1(ADH1A)undergoes histone methylation and promotes the occurrence and development of hepatocellular carcinoma(HCC),which provides new ideas for molecularly targeted diagnosis and treatment of hepatocellular carcinoma.Methods:1.Bioinformatics analysis of the histone methylation status of ADH1 A,and screening of key histone methyltransferases that mediate the histone methylation of ADH1A;q RT-PCR was used to detect the expression of EZH2 in hepatocellular carcinoma tissues,and immunohistochemistry was used to detect the expression of EZH2 in hepatocellular carcinoma and adjacent normal tissues;2.q RT-PCR was used to detect the expression of EZH2 in hepatocellular carcinoma cells(Hep G2,Huh7,PLC/RPF/5,MHCC-9 7H)and normal liver cells(L02);GSK126 histone methylation inhibitor was used to detect the expression changes of ADH1 A at different concentrations;3.Western Blot was used to detect the protein expression level of ADH1 A after knockdown and overexpression of EZH2 in hepatocellular carcinoma;4.Rescue experiments verified whether ADH1 A was involved in the regulation of EZH2 promoting the proliferation of hepatocellular carcinoma;5.q RT-PCR was used to detect lncRNA MPAKPK5-AS1,l ncRNA MIR4435-2HG and LINC00680 were expressed in hepatocellular carcinoma,and their relationship with the prognosis of patients was analyzed;6.Combining bioinformatics methods with FISH and immunofluorescence techniques to analyze the co-localization of lncRNA MAPAKPK5-AS1 and EZH2 in hepatocellular carcinoma cells;7.Western Blot was used to detect the protein expression level of ADH1 A after knocking down lncRNA MAPAKP5-AS1 in hepatocellular carcinoma;8.The rescue experiment verified that lncRNA MAPKAPK5-AS1 regulates the expression of ADH1 A through EZH2 to regulate the proliferation and migration ability of hepatocellular carcinoma.Results:1.Bioinformatics analysis showed that ADH1 A may have histone methylation in hepatocellular carcinoma,and EZH2 is a key gene that mediates ADH1 A histone methylation.2.The overexpression of EZH2 in HCC tissues was significantly negatively correlated with the mRNA and protein expression levels of ADH1 A,and the EZH2+/ADH1 A phenotype was closely related to the poor prognosis of patients(all P <0.05).3.In the liver cancer cells Hep G2 and MHCC-97 H,as the concentration of the histone methylation inhibitor GSK126 increased,the protein expression level of ADH1 A also increased;the protein expression level of ADH1 A also increased significantly after knocking down EZH2,and at the same time The expression of H3K27me3 was decreased;and the protein expression level of ADH1 A was significantly decreased after overexpression of EZH2(all P < 0.05).4.Knockdown of EZH2 in Hep G2 and MHCC-97 H can significantly inhibit the proliferation and migration of hepatocellular carcinoma cells.Rescue experiments have demonstrated that simultaneous knockdown of ADH1 A and EZH2 can reverse the inhibition of EZH2 alone on the migration of hepatocellular carcinoma(P< 0.05).5.Bioinformatics analysis of GEO and TCGA databases showed that lncRNA MAPKAPK5-AS1 was significantly correlated with EZH2 and ADH1 A in hepatocellular carcinoma(both P<0.05).6.The detection and analysis of 60 cases of hepatocellular carcinoma and paired adjacent normal tissues found that lncRNA MAPKAPK5-AS1 was highly expressed in hepatocellular carcinoma tissues,and lncRNA MAPKAPK5-AS1 was significantly positively correlated with EZH2 and significantly negatively correlated with ADH1A(all P < 0.05).7.After knocking down lncRNA MAPKAPK5-AS1 in Hep G2 and MHCC-97 H,the protein expression of ADH1 A was significantly increased.Rescue experiments proved that simultaneous knockdown of ADH1 A and lncRNA MAPKAPK5-AS1 could reverse the effects of knocking out lncRNA MAPKAPK5-AS1 alone on hepatocellular carcinoma Inhibition of proliferation and migration(both P < 0.05).8.FISH and immunofluorescence colocalization proved that lncRNA MAPKAPK5-AS1 and EZH2 co-localized in liver cancer cells,and rescue experiments showed that lncRNA MAPKAPK5-AS1 could regulate the expression of ADH1 A through EZH2(all P <0.05).Conclusions: 1.lncRNA MAPKAPK5-AS1 mediates EZH2 to regulate the expression of ADH1 A and promote the malignant biological behavior of hepatocellular carcinoma in hepatocellular carcinoma.2.Overexpression of lncRNA MAPKAPK5-AS1 and EZH2 predict high aggressiveness of HCC and poor prognosis of tumor patients.