MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway

Dawit Tesfaye,Eva Held,Michael Hoelker,Dessie Salilew Wondim,Ernst Tholen,Christiane Neuhoff,Samuel Gebremedhn,Karl Schellander,Hari Om Pande

doi:10.1186/s13048-018-0410-3

Abstract

BackgroundThe granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches.ResultsThe role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling pathway, granulosa cells were treated with activin A. Activin A treatment increased cell proliferation and downregulation of both miRNA-424/503 members and its target gene, indicated the presence of negative feedback loop between activin A and the expression of miRNA-424/503.ConclusionThis study suggests that the miRNA-424/503 cluster members are involved in regulating bovine granulosa cell proliferation and cell cycle progression. Further, miRNA-424/503 cluster members target the SMAD7 and ACVR2A genes which are involved in the activin signalling pathway.

Highlights

The granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs
silico analysis mothers against decapentaplegic homolog 7 (SMAD7) and activin receptor type 2A (ACVR2A) are the direct targets of the miRNA424/503 cluster members We demonstrated in our previous study that miR-424/ 503 cluster miRNAs were upregulated in the granulosa cells of preovulatory dominant follicles at day 19 of the oestrous cycle
Overexpression of miR-424/503 cluster members promoted granulosa cell proliferation and cell cycle progression accompanied by downregulation of the SMAD7 and ACVR2A mRNA expression level

Summary

Introduction

The granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). The sequential, well-controlled transformation from the primordial to antral follicle stage is the result of the differentiation and proliferation of GCs that provide essential and vital inputs in the form of steroid hormones, cytokines, and paracrine and autocrine factors during the process of follicular development [5,6,7], which is tightly regulated by array of genes [3, 5, 6, 8, 9] that may in turn fine-tuned by microRNAs [10, 11]. Several genes involved in steroidogenesis (CYP17A1, CYP11A1, HSD3B1, STAR), cell proliferation/the cell cycle (CCND2, PCNA), gonadotropin receptors (LHCGR, FSHR) and growth factors (GDF9, BMP2, Activins, IGF1, IGF2) have been found to be altered in granulosa cells depending on the size and stage of follicular development [3, 12,13,14,15,16]

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