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

Regulatory effect of miR-128 on Nkx2-5 gene during the process of rat BMSCs differentiating into cardiomyocyte-like cells

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、22 d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Nkx2-5 mRNA的3'UTR结合情况良好。提取心肌细胞总RNA,扩增Nkx2-5 mRNA 3'UTR片段,构建Nkx2-5 3'UTR-pmir GLO荧光素酶报告载体,双荧光素酶检测转染Nkx2-5 3'UTR-pmir GLO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6 d的Nkx2-5和miR-128的表达量分别设为1,诱导10 d的Nkx2-5表达量为4.33±1.32、22 d为14.78±6.05,诱导10 d的miR-128表达量为0.776 2±0.054 3、22 d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-pmir GLO的荧光素酶表达下降为0.435 4±0.072 6。结论 miR-128通过结合Nkx2-5 mRNA 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。

Objective To investigate the regulatory effect of miR-128 on Nkx2-5 gene during the process of rat bone marrow mesenchymal stem cells （ BMSCs） differentiating into cardiomyocyte-like cells.Methods BMSCs were isolated from bone marrow and induced into cardiomyocyte-like cells using 5-azacytidine.The expression of miR-128 and Nkx2-5 was determined by real-time PCR for the induction of 6, 10, 14, 18 and 22 days.The matching relationships of miR-128 and Nkx2-5 gene were predicted by using bioinformatics method which showed that miR-128 was well combined with 3′-un-translated region （3′UTR） of Nkx2-5 mRNA.The total RNA of the myocardial cells was extracted.We amplified Nkx2-5 mRNA 3 ′UTR segment and constructed Nkx2-5 3′UTR-pmirGLO luciferase reporter plasmid.Luciferase activities of Nkx2-5 3′UTR-pmirGLO, miR-128 mimics or the control miRNA were detected by using the dual-luciferase assay system. The targeted relationships of miR-128 and Nkx2-5 were identified.Results If the expression of miR-128 and Nkx2-5 on the sixth day was arbitrarily defined as 1, the expression of Nkx2-5 mRNA on the tenth day was 4.33 ±1.32 and increased to 14.78 ±6.05 on the twenty-second day.The expression of miR-128 on the tenth day was 0.776 2 ±0.054 3 and de-creased to 0.197 6 ±0.083 6 on the twenty-second day.The expression of miR-128 was negatively correlated with Nkx2-5 （r=-0.546, P=0.021）.The miR-128 decreased the luciferase expression of Nkx2-5 3′UTR-pmirGLO to 0.435 4 ± 0.072 6as compared with that of the control miRNA （which was difined as 1）.Conclusion The miR-128 may inhibit the Nkx2-5 expression by combing with Nkx2-5 mRNA 3′UTR, which negatively regulates the expression of Nkx2-5 during the process of BMSCs differentiating into cardiomyocyte-like cells.