miR-181a对牛骨骼肌卫星细胞分化的影响

Effect of miR-181a on the differentiation of bovine skeletal muscle satellite cells

为了探讨miR-181a对牛骨骼肌卫星细胞分化的影响,试验将分离培养的牛骨骼肌卫星细胞接种在6孔板中,待细胞汇合度为70%时,用PEI转染miR-181a的模拟物miR-181a-M、抑制剂miR-181a-I、模拟物对照miR-181a-M-NC和抑制剂对照miR-181a-I-NC进入牛骨骼肌卫星细胞中,在0,1,2,3天用Western-blot检测肌肉有关分化蛋白MyoD、MyoG和MYH3以及靶基因HOX-A11蛋白的表达量,用免疫荧光法检测细胞肌管的数量。结果表明:在牛骨骼肌卫星细胞中,转染miR-181a模拟物的牛骨骼肌卫星细胞,有关肌肉分化蛋白质如MyoD、MyoG和MYH3的表达量明显增加,细胞肌管数量显著增多,进一步证实miR-181a促进细胞的分化。在双荧光素酶报告系统中,miRNA-181a和HOX-A11基因的3'-UTR结合时荧光素酶活性明显下降,miRNA-181a和突变的3'-UTR结合时荧光素酶活性没有下降。说明HOX-A11是miR-181a的靶基因,miR-181a对牛骨骼肌卫星细胞的分化具有促进作用。

The aim of the present study was to investigate the effect of miR - 181 a on the differentiation of bovine skeletal muscle satellite cells. The bovine skeletal muscle satellite cells were isolated and seeded in six - well plates. When the cell confluence reached 70% , the miR - 181 a - M, miR - 181a - I, miR - 181a - M - NC and miR - 181a - I - NC were transfeet into the bovine skeletal muscle satellite ceils by using the PEI. The expression of relative muscle differentiated proteins MyoD, MyoG, MYH3 and HOX - A11 and the myotube number were detected by Western - blot and mmunofluorescence at the 0, 1 st, 2nd and 3rd day after tranfection. The results showed that the bovine skeletal muscle sat- ellite cells transfected with miR - 181a mimics were significantly increased in the expression of muscle differentiation proteins such as MyoD, MyoG and MYH3, and the number of myocardium transfected with miR - 181a mimics was significantly higher than that of others. It was con- firmed that the cell differentiation could be promoted by the miR - 181a. In the dual luciferase reporter system, when the 3＇- UTR of the HOX -A11 geue was bound to miRNA- 181a, the luciferase activity was significantly decreased. But the luciferase activity did not decrease when the mutated 3＇- UTR was bound to miRNA - 181a. The results further indicated that the HOX - All was the target gene of miR - 181a. In general, the miR -181a could play a role in promoting the differentiation of bovine skeletal muscle satellite cells.