miR-17-3p靶向KCTD15调控延边黄牛前体脂肪细胞分化

miR-17-3p Regulates Preadipocyte Differentiation by Targeting KCTD15 in Yanbian Yellow Cattle

旨在探究miR-17-3p是否可以通过靶向KCTD15调节延边黄牛前体脂肪细胞的分化。本研究使用生物信息学软件鉴定miR-17-3p的同源性,预测miR-17-3p的靶基因;通过在延边黄牛的前体脂肪细胞中转染miR-17-3p mimic或miR-17-3p inhibitor及阴性对照实现细胞内miR-17-3p过表达或抑制表达;采用双荧光素酶报告基因检测系统确定miR-17-3p与KCTD15的靶标关系;使用qRT-PCR和Western blot技术在mRNA和蛋白水平上检测miR-17-3p对KCTD15基因以及脂代谢标志基因PPARγ和C/EBPα表达水平的影响。结果表明,生物信息学软件预测KCTD15可作为miR-17-3p的靶基因,且miR-17-3p在哺乳动物中具有较高的同源性;转染miR-17-3p mimic后脂代谢标志基因PPARγ和C/EBPα的mRNA和蛋白表达量显著或极显著高于转染NC-mimic的对照组(P<0.05或P<0.01);抑制miR-17-3p的表达后,PPARγ和C/EBPα的表达量则显著或极显著低于对照组(P<0.05或P<0.01);双荧光素酶报告基因验证分析显示,过表达miR-17-3p极显著抑制了含有KCTD153′UTR片段的载体的荧光活性(P<0.01);过表达miR-17-3p也会显著或极显著抑制候选靶基因KCTD15 mRNA和蛋白质的表达量(P<0.05或P<0.01);而抑制miR-17-3p的表达则会显著提高靶基因KCTD15的mRNA和蛋白质的表达量(P<0.05)。这些结果说明,miR-17-3p可能通过抑制KCTD15的表达促进延边黄牛前体脂肪细胞分化。

The aim of this study was to explore whether miR-17-3p could target KCTD15 to regulate the preadipocytes differentiation of Yanbian Yellow cattle.Bioinformatics softwares were used to identify homology and predict target genes of miR-17-3p.miR-17-3p mimic or miR-17-3p inhibitor and their negative control were transfected into bovine preadipocytes to overexpress or inhibit the expression of miR-17-3p.The binding sites of miR-17-3p to KCTD15 were verified via double luciferase report system.qRT-PCR and Western blot were used to detect the effect of miR-17-3p on the expressions of PPARγ,C/EBPαand KCTD15 both at mRNA and protein levels.Bioinformatics software prediction showed that KCTD15 was the target gene of miR-17-3p,and miR-17-3p had high homology in mammals.The mRNA and protein expressions of adipogenic marker genes PPARγand C/EBPαafter transfection of miR-17-3p mimic were significantly or extremely significantly higher than those of the control group transfected with NC-mimic(P<0.05 or P<0.01).After the expression of miR-17-3p was inhibited,the expressions of PPARγand C/EBPαwere significantly or extremely significantly lower than that in the control group(P<0.05 or P<0.01).Through the dual-luciferase reporter assay verification,the overexpression of miR-17-3p extremely significantly inhibited the fluorescent activity of the luciferase reporter gene vector containing the 3′UTR fragment of KCTD15(P<0.01).Overexpression of miR-17-3p could also significantly or extremely significantly inhibit the expression of KCTD15 mRNA and protein(P<0.05 or P<0.01).However,inhibition of miR-17-3p expression could significantly increase the expression of KCTD15 mRNA and protein(P<0.05).These results suggest that miR-17-3p may regulate the differentiation of preadipocytes in Yanbian Yellow cattle by inhibiting the expression of KCTD15.