miR-34a Induces Early Apoptosis of Porc-ne Ovarian Granulosa Cells by Activating lncRNA NORHA Transcription
[Objective] miR-34a has been shown to be a pro-apoptotic factor in porcine ovarian granulosa cells (GCs) in our previous study. The aim of this study was to explore the RNA activation mechanism by which miR-34a induced apoptosis in the nucleus, so as to provide a basis for uncovering the molecular mechanism of porcine follicular atresia, and to screen small nucleic acid regulator that regulated procine reproduction. [Method] Bioinformatics methods were used to analyze the characteristics of the porcine miR-34a, the binding sites of miR-34a to the promoter region of target 1ncRNA NORHA, and the binding capacity. The subcellular localization of miR-34a was analyzed in porcine GCs by using nucleocytoplasmic fractionation. The effects of miR-34a on the expression of NORHA,apoptosis marker genes BCL-2 and BAX in porcine GCs were determined by qPCR.Luciferase assay was used to verify the regulatory relationship of miR-34a on the transcriptional activity of the NORHA promoter.Chromatin immunoprecipitation(ChIP)was utilized to detect the enrichment of AGO2 and histone modifiers at the miRNA response element(MRE)in the NORHA promoter in porcine GCs by miR-34a.The regulatory effect of miR-34a on downstream factor FOXO1 expression and GC apoptosis via NORHA were analyzed by western blot and flow cytometry.[Result]The porcine miR-34a was an intergenic miRNA,and its sequence was highly conserved among mammals.Subcellular localization analysis showed that miR-34a was distributed in both nucleus and cytoplasm in porcine GCs.Bioinformatic analysis revealed that the MRE of miR-34a was located at-194 nt--173 nt in the NORHA promoter,and the binding free energy of the two compounds was-23.9 kcal·mol-1.qPCR showed that miR-34a significantly upregulated NORHA expression in porcine GCs.Luciferase assay revealed that miR-34a significantly upregulated the activity of the reporter vector with NORHA promoter,whereas it had no significant effect on the activity of promoter with the mutated MRE of miR-34a.ChIP assay showed that miR-34a increased the enrichment of AGO2,a core member of RNA induced transcriptional activation(RITA)complex,and H3K4me3,a transcription activated histone modifier,while or decreased the enrichment of H3K9me3,a transcription repressed histone modifier at MRE motif of miR-34a in the NORHA promoter.Co-transfection assay showed that knockdown of NORHA significantly reversed the upregulation of FOXO1 protein level and early apoptosis rate caused by miR-34a overexpression,as well as the downregulation of the BCL-2/BAX ratio.[Conclusion]Nuclear miR-34a was an endogenous small activating RNA(saRNA)in porcine GCs,and might be involved in the initiation of porcine follicular atresia by inducing early apoptosis in porcine GCs through target activating the transcription of the pro-apoptotic lncRNA NORHA.
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