| Background:Micro RNAs are small single-stranded non-coding RNAs,which are currently founded to be expressed in virus genome and eucaryon,with length of approximately 18-22 nucleotides.Most micro RNAs degrade or suppress the translation process by complementary binding to the 3'untranslated regions(3'UTR)of the target m RNA,thus regulating the growth and development,cell proliferation,differentiation,apoptosis,metabolism,immune reaction and other biological processes at the post-transcriptional levels.Ischemia/reperfusion,which often happens during the organ transplantation,can result in morbidity and mortality after surgery involving cell death and severe inflammatory reaction.Several studies have reported that micro RNA-191 was dysregulated in liver carcinoma and other liver-related.however,little is known about the function and molecular mechanism of mi R-191 in hepatic ischemia/reperfusion injury.Objective:To explore the function and mechanism of mi R-191 during H/R or in liver tissue during IRI.Methods:First,whether mi R-191 is involved in IRI or H/R was preliminarily analyzed through in vivo and in vitro experiments.WT mice were used to establish the IRI model to detect the expression changes of mi R-191 in ischemic liver tissue.Meanwhile,human liver cell line LO2 H/R model was established to detect the expression level of LO2 mi R-191 at different time points.Then the regulatory effect of mi R-191 on the liver tissue during IRI and H/R was analyzed.After the IRI model was established,the levels of alanine aminotransferase(ALT),Aspartate aminotransferase(AST)and inflammatory factors in ischemic liver tissues of WT and mi R-191 KO mice were detected,and the effects of mi R-191 on liver injury were compared in vivo by HE staining.The expression of mi R-191 in LO2 cells was modulated in vitro and an H/R model was established to analyze the effect of mir-191 on liver cell apoptosis and cell cycle by flow cytometry.Furthermore,the molecular mechanism by which mir-191 regulating IRI and H/R injury was discussed.Firstly,upstream transcription factors that regulate the expression of mir-191 in this process were searched:Transcription factors that may bind to mir-191 in IRI and H/R were predicted by bioinformatic method,and Ch IP assay and Luciferase assay were combined to confirm transcription factors that regulate the expression of mi R-191 in IRI and H/R.Then downstream targets regulated by mi R-191 were searched:Bioinformatics was used to predict possible targets of mir-191,and real-time PCR and luciferase reporter gene were utilized to verify the negative regulation of mir-191 on relevant targets in 293 T cell.Finally,in IRI mice and H/R cells,protein western blot was used to confirm the negative regulation of mir-191 on related targets and the effect of related targets on this process.Through the recovery experiment,the H/R hepatocyte model was established to study the specific signaling pathway of mir-191 by which regulated the downstream targets,thereby affecting the hepatocyte cycle,apoptosis and other phenotypes,so as to regulate the IRI and the H/R injury of hepatocytes.Results:1.The expression of mi R-191 is increased during H/R or IR model.2.Mi R-191 deficiency alleviates cell death and protects the liver from IRI injury.3.MiR-191 overexpression promotes G0/G1 cell cycle arrest and cell apoptosis.4.MiR-191 directly suppresses ZONAB.5.ZONAB is the major downstream target of miR-191 in H/R induced damage.6.Cyclin D1 is a downstream factor of ZONAB in the mediation of mi R-191-induced cell death under hypoxic or ischemic conditions.Conclusion:Under IRI or H/R stress,mi R-191 is markedly upregulated via HIF1?.mi R-191 represses ZONAB/Cyclin D1 signaling to facilitate cell cycle arrest and induce cell death,and subsequently functions in IR or H/R induced cell damage. |
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