miR-1306-5p对肿瘤细胞的增殖具有促进作用,对脂肪细胞增殖与分化的影响尚不明确.课题组前期采集玫瑰冠鸡(Gallus gallus)和科宝鸡胚胎期第8天胸肌组织进行高通量测序,本研究选择测序数据中筛选出的差异表达的miR-1306-5p,旨在探究miR-1306-5p在鸡前脂肪细胞增殖与分化过程中的功能.利用Targetscan和miRanda软件预测miR-1306-5p的靶基因,以脂肪代谢相关基因—骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)为靶基因构建野生型和突变型载体,与miR-1306-5p模拟物共转染鸡成纤维细胞,利用荧光素酶报告基因检测系统验证二者的靶标关系.以miR-1306-5p模拟物和抑制物分别转染鸡前脂肪细胞,利用CCK-8(Cell Counting Kit-8)试剂盒检测细胞增殖能力,通过油红O染色检测油酸诱导的分化情况.结果显示,BMP2野生型与miR-1306-5p模拟物共转染组的荧光素酶活性较阴性对照极显著下降(P＜0.01),而突变型没有显著变化;miR-1306-5p模拟物转染鸡前脂肪细胞48、72和96 h,细胞增殖能力极显著低于阴性对照(P＜0.01);在鸡前脂肪细胞的油酸诱导分化过程中,脂肪酸结合蛋白 4(fatty acid binding protein 4,FABP4)基因表达量极显著升高(P＜0.01),而内源性 miR-1306-5p 和BMP2在诱导分化48和72 h的基因表达量极显著降低(P＜0.01);在鸡前脂肪细胞中转染miR-1306-5p模拟物后脂滴较对照小,转染miR-1306-5p抑制物后脂滴增多.因此,miR-1306-5p可能通过靶向BMP2而抑制鸡前脂肪细胞的分化.本研究为深入探讨miR-1306-5p在鸡前脂肪细胞分化中的作用机制提供基础资料.
Effect of MiR-1306-5p on the Proliferation and Differentiation of Chicken(Gallus gallus)Preadipocytes
The functional studies of miR-1306-5p are mostly found to affect the proliferation of smooth muscle cells and various tumor cells.In the early stage,the research group collected the pectoral muscles of rose-crowned chickens(Gallus gallus)and Kebao chickens on the 8th day of embryonic stage for high-throughput sequencing.In this study,differentially expressed miR-1306-5p was selected from the previous sequencing data.The aim was to investigate the function of miR-1306-5p in the proliferation and differentiation of chicken preadipocytes.Using Targetscan and miRanda software predicted target genes of miR-1306-5p.Select the target gene related to fat,bone morphogenetic protein 2(BMP2),and design the wild type and mutant vector of the target gene BMP2.The vector and miR-1306-5p mimics were constructed to co-transfect chicken fibroblasts,and the target relationship between them was verified by luciferase reporter gene detection system.miR-1306-5p mimics and inhibitors were transfected into chicken preadipocytes,respectively.Cell proliferation capacity was detected by Cell Counting Kit-8(CCK-8)kits.The differentiation induced by oleic acid was detected by oil red O staining.The results showed that the luciferase activity in the BMP2 wild type co-transfected with miR-1306-5p mimics group was extremely significantly decreased compared with the negative control group(P＜0.01),while the mutant type had no significant change.After transfection with miR-1306-5p mimics for 48,72 and 96 h,the proliferation capacity of chicken preadipocytes was extremely significantly lower than that of the negative control group(P＜0.01).The gene expression of fatty acid binding protein 4(FABP4)was extremely significantly increased during oleic acid-induced differentiation of chicken preadipocytes(P＜0.01).The gene expressions of endogenous miR-1306-5p and BMP2 were extremely significantly decreased at 48 and 72 h after induction of differentiation(P＜0.01).Lipid droplets were smaller after transfection of miR-1306-5p mimics and increased after transfection of miR-1306-5p inhibitor in chicken preadipocytes.Therefore,miR-1306-5p may inhibit the proliferation and differentiation of chicken preadipocytes by targeting BMP2.This study provides basic data for further exploring the mechanism of miR-1306-5p in chicken preadipocyte differentiation.
Chicken preadipocytesmiR-1306-5pBone morphogenetic protein 2(BMP2)Cell proliferationCell differentiation
国家科技期刊平台
NSTL
万方数据
