Mechanism Study Of MiR-214 Activating The Ferroptosis Driver Genes And Promoting Ferroptosis Sensitivity In Alcoholic Liver Disease

Objective:miR-214 is significantly up-regulated in the liver tissue of alcoholic hepatitis patients.By studying the positive regulatory effect of miR-214 on ferroptosis driver genes,the toxic mechanism of miR-214 on alcohol-exposed hepatocytes was illustrated,which provides new theoretical basis and experimental support for the functional role of miRNA in the pathogenesis of alcoholic liver disease.Methods:The expression data of miR-214 in alcoholic hepatitis patients and healthy individuals was extracted from GEO database;CCK8 and LDH assays were used to study the toxic effects of miR-214 on alcohol-exposed hepatocytes.The effect of miR-214 on ferroptosis sensitivity of hepatocytes was investigated by miR-214 mimics transfection combining with GPX4 inhibitor RSL3 treatment or GPX4 si RNA transfection.By integrating Target Scan database,mir DIP database,RNAhybrid algorithm and Pearson correlation analysis,the targeting of miR-214 to ferroptosis driver genes and ferroptosis suppressor genes was predicted.Real-time Quantitative PCR and Western Blot experiments were used to verify the regulatory effect of miR-214 on targeted ferroptosisrelated genes.The cellular location of miR-214 in hepatocytes was determined by nuclear/cytosolic fractionation assay.The effects of miR-214 on the stability of mature transcripts and precursor transcripts of targeted genes were investigated by actinomycin D experiments.Chromatin Immunoprecipitation assay(Ch IP)was used to analyze miR-214-mediated chromatin activation and RNA polymerase II enrichment at the promoter region of targeted gene.Finally,the AGO proteins involved in the regulatory effects of miR-214 were identified by RNA immunoprecipitation assay(RIP)and co-transfection experiments.Results:In this study we found that the expression of miR-214 was significantly increased in the liver of alcoholic hepatitis patients.miR-214 can further inhibit the cell activity of alcohol-exposed hepatocytes and significantly increase the levels of released LDH.In addition,we also found that miR-214 can significantly increase the sensitivity of hepatocytes to ferroptosis.In silico analyses and cellular experiments showed that miR-214 targeted ferroptosis driver genes ACSL4,SLC38A1,and PRKAA2,and promoted their expressions.,while miR-214 has no regulatory effect on ferroptosis suppressor genes.Nuclear/cytosolic fractionation assay showed that miR-214 was mainly distributed in the nucleus,and miR-214 could significantly increase the pre-m RNA expressions of target genes,and had no effects on the stability of mature transcripts and precursor transcripts.Chromatin immunoprecipitation(Ch IP)assay showed that miR-214 could significantly increase the enrichment of H3K27 ac,H3K4me1,and RNA polymerase II at the promoter region of target genes.The results of RNA immunoprecipitation(RIP)assay showed that miR-214 bound AGO2 protein but not AGO1 protein in the nucleus,but had no interaction with AGO1 or AGO2 proteins in the cytoplasm.Subsequently,co-transfection experiments of AGO2 si RNA and miR-214 mimics further indicated that the regulatory effect of miR-214 was dependent on the participation of AGO2 protein.Conclusions:In alcoholic liver disease,miR-214 positively regulates the expressions of ferroptosis driver genes,ACSL4,SLC38A1,and PRKAA2,at the transcriptional level in an AGO2-dependent manner,thereby increasing the sensitivity of hepatocytes to ferroptosis.