调控牛CEBPA基因表达的miRNA筛选及对肌内前体脂肪细胞增殖和分化的影响

Screening of miRNA Regulating the Expression of Cattle CEBPA Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Precursor Adipocytes

旨在筛选并验证调控牛增强子结合蛋白A(CCAAT/enhancer binding protein alpha,CEBPA)表达的miRNA及其对肌内前体脂肪细胞增殖和分化的影响。本研究综合运用多个在线软件筛选与CEBPA基因具有潜在调控关系的miRNA,运用qRT-PCR技术检测CEBPA基因在关岭牛各类组织中的表达水平;构建CEBPA-3′UTR-WT和CEBPA-3′UTR-MUT双荧光素酶报告载体,通过双荧光素酶报告系统验证候选miRNA与CEBPA基因的结合关系;分离培养关岭牛肌内前体脂肪细胞,利用qRT-PCR、Western blot、油红O染色和CCK-8探究调控CEBPA的miRNA对牛肌内前体脂肪细胞分化与增殖的影响。生物信息学分析发现,bta-miR-23a、bta-miR-23b-3p、bta-miR-181a、bta-miR-181b、bta-miR-181c、bta-miR-181d与CEBPA基因具有潜在结合关系;qRT-PCR结果表明,CEBPA基因在关岭牛内脏组织、肌肉组织、脂肪、下丘脑以及部分生殖器官中均有表达,在2岁牛背最长肌的表达量极显著高于1日龄犊牛(P<0.01);双荧光素酶报告系统结果表明,bta-miR-23a、bta-miR-23b-3p、bta-miR-181a能直接结合牛CEBPA基因3′UTR;细胞试验结果表明,CEBPA基因在牛肌内前体脂肪细胞分化中的表达水平随时间逐渐升高,bta-miR-23a、bta-miR-23b-3p、bta-miR-181a在分化0 d表达水平极显著高于其他时期(P<0.01),整个分化时期bta-miR-23b-3p、bta-miR-181a与CEBPA基因表达趋势相反;过表达miR-23a、miR-23b-3p、miR-181a均能极显著降低CEBPA基因mRNA和蛋白表达量(P<0.01),并降低牛肌内前体脂肪细胞中脂滴含量;bta-miR-23a、bta-miR-23b-3p、bta-miR-181a能阶段性促进细胞增殖。结果表明,bta-miR-23a、bta-miR-23b-3p、bta-miR-181a能阶段性的促进牛肌内前体脂肪细胞增殖,并通过直接负调控CEBPA的表达来间接影响该细胞分化。

To screen and verify the miRNA regulating the expression of cattle CEBPA (CCAAT/enhancer binding protein alpha, CEBPA) and their effects on the proliferation and differentiation of intramuscular precursor adipocytes. In this study, multiple online softwares were used to screen miRNA with potential regulation relationships with CEBPA genes. qRT-PCR technology was used to detect CEBPA gene expression levels in various tissues of Guanling cattles. By constructing CEBPA -3′UTR-WT and CEBPA -3′UTR-MUT dual luciferase reporter vectors, and then it was used to verify the regulatory relationship between candidate miRNAs and CEBPA genes. After Guanling cattle intramuscular preadipocytes were isolated and cultured, qRT-PCR, Western blot, Oil Red O staining and CCK-8 were used to explore the effect of miRNA regulating CEBPA on the differentiation and proliferation of bovine intramuscular preadipocytes. Bioinformatics analysis revealed that bta-miR-23a, bta-miR-23b-3p, bta-miR-181a, bta-miR-181b, bta-miR-181c, bta-miR-181d and CEBPA genes had potential regulating relationship. CEBPA gene was expressed in the visceral tissues, muscle tissues, fat, hypothalamus and part of the reproductive organs of Guanling cattles, the expression level in the longissimus dorsi of 2-year-old cattle was significantly higher than that in 1 day old calf ( P <0.01). The results of the dual luciferase reporter carrier system indicated that bta-miR-23a, bta-miR-23b-3p, and bta-miR-181a could regu- lation the cattle CEBPA gene 3′UTR;Cell test results showed that the expression level of CEBPA gene in the differentiation of cattle intramuscular precursor adipocytes gradually increased with time, and bta-miR-23a, bta-miR-23b-3p and bta-miR-181a showed extremely significantly higher expression levels at 0 d differentiation than other periods ( P <0.01), the expression trends of bta-miR-23b-3p and bta-miR-181a were opposite with CEBPA gene during the whole differentiation period;Overexpression of miR-23a, miR-23b-3p, miR-181a could extremely significantly reduce CEBPA gene mRNA and protein expression ( P <0.01), and reduce the content of lipid droplets in cattles intramuscular precursor adipocytes. bta-miR-23a, bta-miR-23b-3p, bta-miR-181a could promote cell proliferation in stages. The results indicate that bta-miR-23a, bta-miR-23b-3p, bta-miR-181a can promote the proliferation of intramuscular precursor adipocytes of cattles in stages, and indirectly affect the differentiation of this cell by directly negatively regulating the CEBPA expression.