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

doi:10.11843/j.issn.0366-6964.2023.12.017

Abstract

Abstract: 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 NR4A1 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 complex Ⅰ were respectively detected by transfection miR-200b mimic and mimic NC, respectively, and the expression levels of mitochondrial oxidative phosphorylation-related genes were 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 NR4A1 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 ATP6V1G1 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.

Key words: sheep, miR-200b, promoter, follicular granulosa cells, mitochondria

CLC Number:

S826.3

SONG Pengyan, WANG Siwei, YUE Qiaoxian, ZHANG Yinliang, CHEN Xiaoyong, ZHOU Rongyan. Identification of oar-miR-200b Promoter and Effects on Mitochondrial Function in Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5066-5076.

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

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