Conserved miR-26b enhances ovarian granulosa cell apoptosis through HAS2-HA-CD44-Caspase-3 pathway by targeting HAS2.

Jiying Liu,Qifa Li,Honglin Liu,Wang Yao,Fei Tu,Zhuang Xie,Xinyu Li,Zengxiang Pan

doi:10.1038/srep21197

Abstract

The hyaluronan synthase 2 (HAS2)-hyaluronic acid (HA)-CD44-Caspase-3 pathway is involved in ovarian granulosa cell (GC) functions in mammals. HAS2 is a key enzyme required for HA synthesis and is the key factor in this pathway. However, the regulation of HAS2 and the HAS2-mediated pathway by microRNAs in GCs is poorly understood. Here, we report that miR-26b regulates porcine GC (pGC) apoptosis through the HAS2-HA-CD44-Caspase-3 pathway by binding directly to the 3′- untranslated region of HAS2 mRNA. Knockdown of miR-26b reduced pGC apoptosis. Luciferase reporter assays demonstrated that HAS2 is a direct target of miR-26b in pGCs. Knockdown and overexpression of miR-26b increased and decreased, respectively, HA content, and HAS2 and CD44 expression in pGCs. At the same time, inhibition and overexpression of miR-26b decreased and increased the expression of Caspase-3, a downstream factor in the HAS2-HA-CD44 pathway. Moreover, knockdown of HAS2 enhanced pGC apoptosis, reduced the inhibitory effects of a miR-26b inhibitor on pGC apoptosis, repressed HA content and CD44 expression, and promoted Caspase-3 expression. In addition, overexpression of HAS2 has a opposite effect. Collectively, miR-26b positively regulates pGC apoptosis via a novel HAS2-HA-CD44-Caspase-3 pathway by targeting the HAS2 gene.

Highlights

The hyaluronan synthase 2 (HAS2)-hyaluronic acid (HA)-CD44-Caspase-3 pathway is involved in ovarian granulosa cell (GC) functions in mammals
We previously demonstrated that miR-26b is an important epigenetic regulator of porcine GC (pGC) function and follicular atresia[27]
To determine the mechanism by which HAS2 regulates pGC apoptosis, we examined the level of Hyaluronic acid (HA) and expression of CD44 and Caspase-3 in pGCs treated with HAS2-small interfering RNA (siRNA)

Summary

Introduction

The hyaluronan synthase 2 (HAS2)-hyaluronic acid (HA)-CD44-Caspase-3 pathway is involved in ovarian granulosa cell (GC) functions in mammals. We report that miR-26b regulates porcine GC (pGC) apoptosis through the HAS2-HA-CD44-Caspase-3 pathway by binding directly to the 3′- untranslated region of HAS2 mRNA. Knockdown of CCAAT-enhancer-binding protein CEBβ decreases the HAS2 mRNA level and results in the arrest of pGCs at S phase of the cell cycle, indicating that HAS2 plays an important role in GC function[13]. In GCs, follicle-stimulating hormone (FSH) stimulates HAS2 expression[12] and affects downstream events of the HAS2-HA-CD44-Caspase-3 pathway such as HA synthesis, CD44 expression, and Caspase-3 expression and activity[4,17] Transcription factors such as CREB1, RAR, SP1, SP3, and YY1 regulate HAS2 expression by binding to functional binding sites within the HAS2 promoter region[20]. Our findings aid understanding of the epigenetic regulation of HAS2 and elucidation of the miR-26b regulation network

Methods

Results

Conclusion