miR-128-1-5p对绵羊前体脂肪细胞增殖和分化调节作用的研究

doi:10.11843/j.issn.0366-6964.2020.005

摘要/Abstract

摘要： 旨在研究miR-128-1-5p对绵羊前体脂肪细胞增殖与分化的调节作用。本研究经理论预测和试验研究验证miR-128-1-5p的靶基因；过表达miR-128-1-5p后，用qPCR检测增殖标志基因的表达，CCK-8和EdU检测细胞增殖情况；用qPCR和Western blotting检测miR-128-1-5p和其靶基因在前体脂肪细胞分化中的表达趋势；通过过表达或抑制miR-128-1-5p研究其对绵羊前体脂肪细胞分化的调节机制；用油红O染色检测成脂能力。结果表明，KLF2是miR-128-1-5p的靶基因。前体脂肪细胞增殖过程中，过表达miR-128-1-5p后，增殖标志基因的表达量显著或极显著降低（P<0.05或P<0.01），细胞活力显著或极显著降低（P<0.05或P<0.01），新生细胞数显著减少（P<0.05）。前体脂肪细胞分化过程中，miR-128-1-5p与KLF2的表达呈负相关；过表达miR-128-1-5p极显著下调了KLF2 mRNA的表达量（P<0.01），显著下调了KLF2蛋白的表达量（P<0.05），显著或极显著上调了分化标志基因mRNA的表达（P<0.05或P<0.01），产生更多脂滴；抑制miR-128-1-5p则结果相反。综上所述，miR-128-1-5p与KLF2存在结合位点。miR-128-1-5p抑制绵羊前体脂肪细胞的增殖，在分化过程中通过靶向抑制KLF2的表达，促进前体脂肪细胞分化和脂滴沉积。

关键词: 绵羊, 前体脂肪细胞, miR-128-1-5p, KLF2, 增殖, 分化

Abstract: This study aimed to reveal the regulating role of miR-128-1-5p in the proliferation and differentiation of ovine preadipocytes. The target genes of miR-128-1-5p were theoretically predicted and experimentally validated. After overexpressing miR-128-1-5p, the expression of proliferation marker genes were detected by qPCR, and the cell proliferation was examined by CCK-8 and EdU. The expression trends of miR-128-1-5p and its target genes during the differentiation of ovine preadipocytes were detected by qPCR and Western blotting. The regulatory mechanism of miR-128-1-5p on the ovine preadipocyte differentiation was revealed by overexpressing or inhibiting miR-128-1-5p. The cell adipogenic effect was tested by Oil Red O staining. The results showed that KLF2 was a target gene of miR-128-1-5p. During the proliferation of ovine preadipocytes, after miR-128-1-5p was overexpressed, the expressions of proliferation marker genes were significantly or extremely significantly decreased (P<0.05 or P<0.01), the cell vitality was also significantly or extremely significantly decreased (P<0.05 or P<0.01), and the newborn cell number was significantly reduced (P<0.05). During the differentiation of ovine preadipocytes, a negative correlation was found between the expressions of miR-128-1-5p and KLF2; After miR-128-1-5p was overexpressed, the mRNA and protein expression of KLF2 were extremely significantly (P<0.01) and significantly (P<0.05) decreased, respectively; The mRNA expressions of differentiation marker genes were significantly or extremely significantly increased (P<0.05 or P<0.01) in the miR-128-1-5p overexpressing cells; More lipid droplets were deposited; The results were opposite after miR-128-1-5p was inhibited. In conclusion, there is a binding site between miR-128-1-5p and KLF2. miR-128-1-5p inhibits the proliferation of ovine preadipocytes. miR-128-1-5p promotes the differentiation of ovine preadipocytes and lipid droplets deposition by targeting and negatively regulating KLF2 in the process of differentiation.

Key words: sheep, preadipocyte, miR-128-1-5p, KLF2, proliferation, differentiation

中图分类号:

S826.2

刘建华, 梁煜, 车雨彤, 王凤, 刘旭莹, 赵弼时, 王强, 安丽霞, 乔利英, 刘文忠. miR-128-1-5p对绵羊前体脂肪细胞增殖和分化调节作用的研究[J]. 畜牧兽医学报, 2020, 51(6): 1207-1218.

LIU Jianhua, LIANG Yu, CHE Yutong, WANG Feng, LIU Xuying, ZHAO Bishi, WANG Qiang, AN Lixia, QIAO Liying, LIU Wenzhong. Study on the Regulation of miR-128-1-5p in Proliferation and Differentiation of Ovine Preadipocyte[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1207-1218.

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