绵羊miR-200b启动子鉴定及其对卵泡颗粒细胞线粒体功能的影响

Identification of oar-miR-200b Promoter and Effects on Mitochondrial Function in Follicular Granulosa Cells

旨在鉴定绵羊miR-200b的启动子区并研究其对卵泡颗粒细胞线粒体功能的影响。本研究体外分离培养绵羊卵泡颗粒细胞,通过构建包含3个不同长度启动子区重组质粒,利用生物信息软件和双荧光素酶报告试验预测并鉴定绵羊miR-200b核心启动子区;利用在线工具预测转录因子NR4A1的结合位点;通过双荧光素酶报告试验和qRT-PCR检测过表达NR 4A1基因对miR-200b启动子活性和表达水平的影响;通过转染miR-200b mimic和mimic NC,分别检测颗粒细胞线粒体形态和数量、线粒体膜电位、ROS水平、ATP和线粒体呼吸链复合物Ⅰ的浓度,并检测线粒体氧化磷酸化相关基因表达水平。结果表明,miR-200b-P1(-159/+36 nt)活性最高,确定该区域为miR-200b核心启动子区;预测出核心启动子存在1个NR4A1的结合位点,且过表达NR 4A1基因显著上调miR-200b启动子活性和miR-200b的表达水平(P<0.01)。与mimic NC组相比,转染miR-200b mimic导致颗粒细胞平均光密度显著降低(P<0.01),细胞长度显著缩短(P<0.01)、线粒体膜电位下降(P<0.05)、ATP(P<0.05)和线粒体呼吸链体复合物Ⅰ(P<0.01)的浓度降低、ROS水平升高(P<0.01),同时下调ATP 6V1G1和NDUFV 1的表达(P<0.01),上调NOX 4的表达水平(P<0.01)。该研究确定了绵羊miR-200b的核心启动子区(-159/+36 nt),NR4A1正向调控miR-200b的转录,且miR-200b能够影响线粒体氧化磷酸化过程,诱导颗粒细胞线粒体损伤。该研究结果为进一步揭示miR-200b调控绵羊卵泡发育的机制提供了理论依据。

The purpose of this study was to identify the oar-miR-200b promoter and investigate the effects of miR-200b on mitochondrial function in follicular granulosa cells.Sheep ovarian follicular granulosa cells were isolated and cultured in vitro,recombinant plasmids containing 3 different lengths of promoter region were constructed,and bioinformatics software and dual-luciferase reporting assays were used to predict and identify the oar-miR-200b core promoter region.Online tools were used to predict the binding site of transcription factor NR4A1,and the effect of overexpression of NR 4A1 gene on miR-200b promoter activity and expression levels was detected by dual-luciferase reporter assay and qRT-PCR.Mitochondrial morphology and number,mitochondrial membrane potential,ROS levels,the concentration of ATP and mitochondrial respiratory chain complexⅠwere respectively detected by transfection miR-200b mimic and mimic NC,respectively,and the expression levels of mitochondrial oxidative phosphorylation-related genes were expression,as well as the relative expression of mitochondrial OXPHOS-related genes detected.The result indicated that the highest activity of miR-200b-P1(-159/+36 nt)was regarded as the core promoter region of miR-200b.There was one binding site of NR4A1 in the miR-200b core promoter.The miR-200b promoter activity and miR-200b expression increased in NR 4A1 overexpressed cells(P<0.01).Compared with mimic NC group,transfection of miR-200b mimic resulted in a significant decrease in the mean optical density(P<0.01)and a reduction in cell length(P<0.01)of granulosa cells,decreased mitochondrial membrane potential(P<0.05),concentrations of ATP(P<0.05)and mitochondrial respiratory chain complex I(P<0.01),and increased ROS levels(P<0.01),accompany with down-regulating the expression of ATP 6V1G1 and NDUFV1(P<0.01)and up-regulating the expression level of NOX4(P<0.01).This study determined the core promoter region of oar-miR-200b(-159/+36 nt),NR4A1 positively regulated the transcription of miR-200b,and miR-200b might affect the process of mitochondrial OXPHOS and induce mitochondrial damage in granulosa cells.These results provide a theoretical basis for further revealing the mechanism of miR-200b during sheep ovarian follicular development.