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

ZHU Guo-wei; JU Yan-ling; GAO Zhen-yu

doi:10.3969/j.issn.1002-266X.2015.34.001

Abstract：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.