miR-26b介导Smads信号途径参与鹅颗粒细胞增殖分子机制

The poor productive performance of goose has seriously hampered the development of industry. It was proved that the proliferation and apoptosis of granulosa cells (GCs) were closely related to the level of follicle development and laying performance. Our previous experiments have found that miR-26b is involved in FSH mediated goose granulosa cell proliferation, and identify the differentially expressed genes which were significantly enriched in Samds signaling pathway. We hypothesized that miR-26b mediated Smads signaling pathway could regulate the proliferation of GCs cells, and then affect follicular development. In this project, firstly, the effect of miR-26b on Smads signaling pathway regulation will be investigated by adding specific blocking agent. And then a central element of miR-26b targeting Smads signaling pathway will be involved in the molecular mechanism of granulocyte proliferation. Finally, the pri-miR-26b transcriptional regulation to granulosa cell proliferation will be explored by ChIP techniques. The study will reveal the regulation mechanism of miR26b on the proliferation of goose granulosa cells, and provide the basic data for the comprehensive analysis of the molecular mechanism of goose low fecundity.

鹅的产蛋性能低下严重阻碍了产业的发展。研究证实，颗粒细胞的增殖和凋亡又与卵泡发育和产蛋性能高低密切相关。本项目前期研究已发现miR-26b参与FSH介导的鹅卵泡颗粒细胞增殖，并鉴定出其差异表达基因在Samds信号通路显著富集。据此我们假设miR-26b介导Smads信号途径调控GCs细胞增殖，进而影响卵泡发育。为此本项目通过添加特异性阻断剂探讨miR-26b受 Smads 信号途径调控分子机理；重点研究miR-26b 靶向Smads信号途径的中心元件Smad4参与颗粒细胞增殖分子机制；并采用启动子缺失突变和ChIP等技术解析Smad4通过pri-miR-26b转录调控介导颗粒细胞增殖的作用机理。研究将揭示miR26b对鹅颗粒细胞增殖的调控机理，为全面阐明鹅低繁殖力分子机制提供基础资料。

产蛋性能低下是制约鹅产业的发展的重要原因之一。卵泡颗粒细胞的增殖和凋亡与鹅产蛋性能密切相关。前期研究发现，miR-26b-5p参与鹅卵泡颗粒细胞增殖，其差异表达基因在Samds信号通路显著富集。据此我们假设miR-26b-5p介导Smads信号途径调控鹅卵泡细胞的增殖，进而影响鹅产蛋性能。本项目探究了miR-26b-5p对鹅卵泡颗粒细胞增殖的影响，进而阐明miR-26b-5p通过介导TGF-β/SMADs信号通路发挥其生物学功能及转录调控机制。这不仅揭示了miR-26b-5p参与鹅卵泡颗粒细胞增殖的作用机理，同时也为提高鹅产蛋量提供一定理论依据。.1、miR-26b-5p参与鹅卵泡颗粒细胞增殖，促进雌二醇分泌.研究结果发现转染miR-26b-5p mimics后颗粒细胞增殖率显著上升81.2%，转染miR-26b-5p inhibitor后细胞增殖被抑制；过表达miR-26b-5p后抗凋亡基因BCL-2表达量显著升高，促凋亡基因Caspase-3表达量显著降低，而抑制内源性miR-26b-5p表现出相反趋势(P < 0.01)。此外，过表达miR-26b-5p可以显著增加雌二醇水平，降低孕酮水平，而抑制内源性miR-26b-5p后表现出相反的趋势(P < 0.05)。.2、miR-26b-5p靶向INHBB基因促进卵泡颗粒细胞增殖.miR-26b-5p可与INHBB基因的3’-UTR结合并极显著抑制荧光素酶活性(P < 0.01)，而不能抑制SMAD4荧光素酶活性；此外，miR-26b-5p可以显著降低INHBB基因的表达水平和翻译蛋白质水平(P < 0.05)。.3、转录因子SMAD4启动miR-26b-5p转录参与鹅卵泡颗粒细胞增殖调控.为阐明miR-26b-5p参与鹅卵泡颗粒细胞增殖的转录机制，对miR-26b-5p基因启动子区序列发现的3个SMAD4结合位点进行分析。结果发现过表达SMAD4可以极显著增加miR-26b-5p的表达量(P < 0.01)，转染含SBE3片段，其荧光活性显著增强(P < 0.01)，因此SMAD4可作为miR-26b-5p的一种转录因子。.综上所述，miR-26b-5p受转录因子SMAD4转录调控，并通过靶向INHBB介导TGF-β/SMADs信号通路，促进鹅卵泡颗粒细胞的增殖。