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

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、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Nkx2－53′UTR－pmirGLO荧光素酶报告载体，双荧光素酶检测转染Nkx2－53′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0．7762±0．0543、22 d为0．1976±0．0836。 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Nkx2－53′UTR－pmirGLO的荧光素酶表达下降为0．4354±0．0726。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.