MicroRNA-129促进骨髓间充质干细胞向心肌分化的实验研究

MicroRNA-129 Promotes Cardiomyogenesis in Bone Marrow Mesenchymal Stem Cells

目的研究microRNA-129(mir-129)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)向心肌分化过程中的促进作用及其机制。方法分离培养SD大鼠骨髓间充质干细胞,通过慢病毒转染使细胞获得过表达基因分为4组:对照组(MSCs);慢病毒空转组(Lentiviral vectors+MSCs,Lv-MSCs);mir-129单转染组(mir-129-MSCs);mir-129+糖原合成酶激酶(GSK-3β)双转染组(mir-129+GSK-3β-MSCs)。以10μmol/L浓度的5-氮杂胞苷(5-Aza)诱导MSCs向心肌细胞分化后,分别以第1 d、5 d、10 d、15 d和20 d为时间点,采用realtime-PCR检测GATA-4、NKx2.5、MEF-2C的mRNA水平,以第10 d、15 d、20 d为时间点,用蛋白质印迹法(Western blotting)检测肌钙蛋白I(cTnI)、结蛋白(Desmin)、GSK-3β以及磷酸化β-catenin与非磷酸化β-catenin的表达量。结果与对照组比较,随着研究时间点的推移,mir-129单转染组反映心肌分化的相关标记物基因和蛋白水平明显升高,GSK-3β的表达水平则显著降低,非磷酸化β-catenin/磷酸化β-catenin比值升高;当MSCs同时过表达mir-129和GSK-3β时,相关的心肌标记物基因和蛋白均低于mir-129单转染组,非磷酸化β-catenin/磷酸化β-catenin比值明显降低。结论过表达mir-129能够促进MSCs向心肌细胞分化,可能的机制是通过抑制GSK-3β的生成,从而削弱了对β-catenin的磷酸化抑制,后者游离入核开启调控干细胞下游的心肌分化途径。

Abstract： Objective To explore the induction of cardiomyogenesis of microRNA-129 （mir-129） in rat bone marrow mesenchymal stem cells （BM-MSCs） and its mechanism. Methods BM-MSCs were isolated from Sprague-Dawley rats and cultured in vitro. Overexpression of mir- 129 or both mir- 129 and glycogen synthase kinase-313 （GSK-313 ） in BM-MSCs was produced with a lentiviral vector system. All the BM-MSCs were divided into four groups： control group （MSCs）, Lentiviral vectors ＋ MSCs group （Lv-MSCs）, mir-129 transfection group （mir-129-MSCs）, and mir-129 ＋ GSK-313 double transfection group （mir- 129 ＋ GSK-313-MSCs ）. Five-Azacytidine （ 5 -Aza） （ 10 ~tmol/L ） was used to induce BM-MSCs differentiation into cardiomyocytes. On the 1 st, 5 th, 10 th, 15 th and 20 th day after induction, realtime-PCR was performed to detect mRNA levels of GATA-4,Nkx2.5 and MEF-2C. On the 10 th, 15 th and 20 th day after induction,Western blotting was performed to examine expression levels of cTnl, Desmin, GSK-313, phosphorylated 13-catenin and dephosphorylated 13-catenin. Results Compared with the control group, at respective time points, mRNA levels of cardiomyogenic genes and expression levels of cardiomyocyte-related proteins of mir-129 transfection group were significantly elevated, the expression level of GSK-313 was significantly decreased, and the ratio of dephosphorylated/phosphorylated 13-catenin was significantly elevated. When both mir-129 and GSK-3β were overexpressed in BM-MSCs, mRNA levels of cardiomyogenic genes and expression levels of cardiomyocyte-related proteins were significantly lower than those of mir-129 transfection group, and the ratio of dephosphorylated/phosphorylated 13-catenin was significantly decreased. Conclusion Overex- pression of mir-129 can promote cardiomyogenesis of rat BM-MSCs possibly via inhibiting GSK-3β production and thusdecreasing the inhibition of phosphorylation of 13-catenin which then enters the nucleus and activates downstream signaling pathways that regulate cardiomyogenic differentiation of BM-MSCs.