【摘要】 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96h，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。更多还原

【Abstract】 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 highthroughput 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 cotransfect 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.更多还原