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。
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 KLF 2 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 KLF 2;After miR-128-1-5p was overexpressed,the mRNA and protein expression of KLF 2 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 KLF 2.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 KLF 2 in the process of differentiation.
作者
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
LIU Jianhua;LIANG Yu;CHE Yutong;WANG Feng;LIU Xuying;ZHAO Bishi;WANG Qiang;AN Lixia;QIAO Liying;LIU Wenzhong(College of Animal Science and Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2020年第6期1207-1218,共12页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31972560)
山西省“1331工程”重点学科建设计划(J201811301)。
