MicroRNA 195-5p Targets Foxo3 Promoter Region to Regulate Its Expression in Granulosa Cells.

Yinshan Bai,Bo Pan,Xiaoshu Zhan,Julang Li,Hailey Silver

doi:10.3390/ijms22136721

Abstract

Forkhead box O3 (Foxo3) is a member of the FOXO subfamily within the forkhead box (FOX) family, which has been shown to be essential for ovarian follicular development and maturation. Previous studies have shown the abundant expression of miR-195-5p in the nuclei of porcine granulosa cells (GCs), suggesting its potential role during ovarian follicle growth. In this study, a conditional immortalized porcine granulosa cell (CIPGC) line was used to determine whether the expression of Foxo3 could be regulated by the nuclear-enriched miR-195-5p. Through silico target prediction, we identified a potential binding site of miR-195-5p within the Foxo3 promoter. The over-expression of miR-195-5p increased Foxo3 expression at both mRNA and protein levels, while the knockdown of miR-195-5p decreased the expression of Foxo3. Furthermore, driven by the Foxo3 promoter, luciferase reporter activity was increased in response to miR-195-5p, while the mutation of the miR-195-5p binding site in the promoter region abolished this effect. In addition, the siRNA knockdown of Argonaute (AGO) 2, but not AGO1, significantly decreased Foxo3 transcript level. However, miR-195-5p failed to upregulate Foxo3 expression when AGO2 was knocked down. Moreover, chromatin immunoprecipitation (CHIP) assay showed that anti-AGO2 antibody pulled down both AGO2 and the Foxo3 promoter sequence, suggesting that AGO2 may be required for miR-195-5p to regulate Foxo3 expression in the nucleus. Additionally, Foxo3 expression was significantly increased by valproic acid (VPA), the inhibitor of deacetylase, as well as by methyltransferase inhibitor BIX-01294, indicating the involvement of histone modification. These effects were further enhanced in the presence of miR-195-5p and were decreased when miR-195-5p was knocked down. Overall, our results suggest that nuclear-enriched miR-195-5p regulates Foxo3 expression, which may be associated with AGO2 recruitment, as well as histone demethylation and acetylation in ovarian granulosa cells.

Highlights

IntroductionMicroRNA (miRNA) have been traditionally known to post-transcriptionally silence gene expression in the cytoplasm via binding to the 30 -UTR of mRNA [1]
Nuclear and cytoplasmic components were isolated from conditional immortalized porcine granulosa cell (CIPGC), and the separation of the two components was confirmed via Western blot analysis, using anti-LAMIN B and anti-GAPDH antibodies to target nuclear and cytoplasmic-specific proteins, respectively (Figure 1A)
Forkhead box O3 (Foxo3) exerts its transcriptional activity in the nucleus to suppress follicular growth in quiescent primordial follicles [25]

Summary

Introduction

MicroRNA (miRNA) have been traditionally known to post-transcriptionally silence gene expression in the cytoplasm via binding to the 30 -UTR of mRNA [1]. It has been revealed that some miRNAs are enriched in the cell nucleus, where they regulate gene expression at the transcriptional level [2,3]. Argonaute (AGO) 2 binds to and unwinds the double-stranded small RNA in the cytoplasm, creating mature miRNA [4]. The miRNA-AGO2 complex may subsequently be transported into the nucleus via Importin

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