朱国伟，鞠延玲，高振宇(锦州市中心医院，辽宁锦州121001)

大鼠BMSCs向心肌样细胞分化过程中miR-128对Nkx2-5基因的调控作用

朱国伟，鞠延玲，高振宇
(锦州市中心医院，辽宁锦州121001)

摘要:目的探讨大鼠骨髓间充质干细胞(BMSCs)向心肌样细胞分化过程中miR-128对Nkx2-5基因的调控作用。方法分离培养大鼠BMSCs，应用5-氮杂胞苷将其诱导为心肌样细胞，采用Real-time PCR法检测诱导6、10、14、18、22d时miR-128、Nkx2-5基因的表达。用生物信息学方法对miR-128和Nkx2-5基因的靶向匹配关系进行预测，显示miR-128与Nkx2-5mRNA的3'UTR结合情况良好。提取心肌细胞总RNA，扩增Nkx2-5mRNA 3'UTR片段，构建Nkx2-5 3'UTR-pmirGLO荧光素酶报告载体，双荧光素酶检测转染Nkx2-5 3'UTR-pmirGLO以及miR-128模拟物或对照miRNA的荧光强度，鉴定miR-128与Nkx2-5的靶向关系。结果将诱导6d的Nkx2-5和miR-128的表达量分别设为1，诱导10d的Nkx2-5表达量为4.33±1.32、22d为14.78±6.05，诱导10d的miR-128表达量为0.776 2±0.054 3、22d为0.197 6±0.083 6。miR-128与Nkx2-5的表达呈负相关(r =－0.546，P =0.021)。相对于转染对照miRNA(设定其荧光素酶活性为1)，转染miR-128能使Nkx2-5 3'UTR-pmirGLO的荧光素酶表达下降为0.435 4±0.072 6。结论miR-128通过结合Nkx2-5mRNA 3'UTR抑制其表达，具有负性调控BMSCs向心肌样细胞分化过程中Nkx2-5表达的作用。

关键词:微小核糖核酸; Nkx2-5基因;骨髓间充质干细胞;心肌样细胞;大鼠

Regulatory effect ofmiR-128 on Nkx2-5 geneduring the process of rat BMSCsdifferentiating into cardiomyocyte-like cells

ZHU Guo-wei，JU Yan-ling，GAO Zhen-yu
(Jinzhou Central Hospital，Jinzhou 121001，China)

Abstract:Objective To investigate the regulatory effect ofmiR-128 on Nkx2-5 geneduring the process of rat bonemarrowmesenchymal stem cells(BMSCs)differentiating into cardiomyocyte-like cells.Methods BMSCs were isolated from bonemarrow and induced into cardiomyocyte-like cells using 5-azacytidine.The expression ofmiR-128 and Nkx2-5 wasdetermined by real-time PCR for the induction of 6，10，14，18 and 22days.Thematching relationships ofmiR-128 and Nkx2-5 gene were predicted by using bioinformaticsmethod which showed thatmiR-128 was well combined with 3'-untranslated region(3'UTR)of Nkx2-5mRNA.The total RNA of themyocardial cells was extracted.We amplified Nkx2-5mRNA 3 'UTR segment and constructed Nkx2-5 3' UTR-pmirGLO luciferase reporter plasmid.Luciferase activities of Nkx2-5 3'UTR-pmirGLO，miR-128mimics or the controlmiRNA weredetected by using thedual-luciferase assay system.The targeted relationships ofmiR-128 and Nkx2-5 were identified.Results If the expression ofmiR-128 and Nkx2-5 on the sixthday was arbitrarilydefined as 1，the expression of Nkx2-5mRNA on the tenthday was 4.33±1.32 and increased to 14.78±6.05 on the twenty-secondday.The expression ofmiR-128 on the tenthday was 0.776 2±0.054 3 anddecreased to 0.197 6±0.083 6 on the twenty-secondday.The expression ofmiR-128 was negatively correlated with Nkx2-5(r =－0.546，P = 0.021).ThemiR-128decreased the luciferase expression of Nkx2-5 3'UTR-pmirGLO to 0.435 4± 0.072 6as compared with that of the controlmiRNA(which wasdifined as 1).Conclusion ThemiR-128may inhibit the Nkx2-5 expression by combing with Nkx2-5mRNA 3'UTR，which negatively regulates the expression of Nkx2-5during the process of BMSCsdifferentiating into cardiomyocyte-like cells.

Key words:microRNA; Nkx2-5 gene; bonemarrowmesenchymal stem cells; cardiomyocyte-like cells;rats

Nkx2-5基因是心肌细胞发育过程中早期表达的转录因子，于心肌细胞分化前已有表达，开始见于心肌前体细胞并于心肌细胞分化阶段持续表达，在胚胎和成体心肌细胞中稳定表达，是调控心肌细胞分化的重要转录因子［1～3］。微小RNA(miRNA)是一类小的内源性非编码RNA，广泛参与基因表达的调控，但miRNA参与心肌样细胞的诱导分化的报道少见。2014年3月，我们应用5-氮杂胞苷诱导大鼠骨髓间充质干细胞(BMSCs)分化为心肌样细胞，检测分化过程中miR-128和Nkx2-5的表达，探讨miR-128对Nkx2-5基因表达的调控作用。……