Mechanism Study Of The Regulatory Roles Of Hsa-miR-1301-3p In ADH6,ALDH5A1,and ALDH8A1 Production And Alcohol Metabolism

Objective To explore the regulatory roles of miRNAs in the production of alcohol metabolism enzymes,and their potential effects on the process of alcohol metabolism and alcohol induced liver injury.Methods The alcohol-metabolizing enzymes expressed in human liver were selected by screening their mRNA data in 49 paired liver tissues from The Cancer Genome Atlas(TCGA)database.The miRNAs potentially targeting alcohol metabolizing genes were predicted using the miRTar.human database,and the free energy of miRNA:mRNA duplexes was calculated by RNAhybrid algorithm,respectively.The correlations between alcohol metabolism genes and miRNAs were calculated by Pearson correlation analysis,based on their RNA levels in liver tissues from TCGA database.Candidate miRNA was selected to evaluate our bioinformatic predictions using a series of biochemical assays.Fluorescent-based RNA electrophoretic mobility shift assay(FREMSA)and luciferase reporter gene assay were used to detect the binding status between miRNA and its targeting site.Furthermore,qRT-PCR and Western blotting experiments were carried out after cell transfection of miRNA mimics to observe the regulatory effects of exogeneous miRNA on its endogenous target genes expression.Finally,the regulatory roles of miRNA in the target gene production was tested in HepG2 and Huh7 cells after mi RNA mimics transfection and alcohol treatment.Results Our results showed that 11 miRNAs might target ADH and ALDH genes in human liver.The miRNA hsa-miR-1301-3p was selected for focused experiments to test the reliability of predictions made using bioinformatics because the expression of hsa-miR-1301-3p in liver exhibited the highest inverse correlations with mRNAs for ALDH8A1(r = –0.480),ADH6(r = –0.469),and ALDH5A1(r = –0.459).FREMSAs and luciferase reporter gene assays showed the direct interactions between hsa-miR-1301-3p and its cognate mRNA targets.Mi RNA mimics transfection assays showed that exogeneous hsa-miR-1301-3p suppressed endogenous ADH6,ALDH5A1 and ALDH8A1 production in hepatic cells.Further,hsa-miR-1301-3p was able to suppress ethanol-dependent induction of ADH6,ALDH5A1 and ALDH8A1 expression.Conclusion We identified hsa-miR-1301-3p as a new epigenetic factor involved in the regulation of the alcohol-aldehyde-acetate metabolism pathway by suppression of ADH6,ALDH5A1 and ALDH8A1.