مقالات پذیرفته شده در سومین کنگره بین المللی زیست پزشکی
Transient induction of Cdk9 is critical is the early stage of myocardial differentiation
Transient induction of Cdk9 is critical is the early stage of myocardial differentiation
Vahideh Tarhriz,1,*Shirin Eyvazi,2Soheila Montazersaheb,3Hossein Ghanbarian,4
1. Molecular Medicine Research Center, Biomedicine institute, Tabriz University of Medical Sciences, Tabriz, Iran 2. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3. Molecular Medicine Research Center, Biomedicine institute, Tabriz University of Medical Sciences, Tabriz, Iran 4. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Introduction: Muscle development (differentiation) is a multi‐step pathway. Myogenic regulatory factors (MRFs) and also small noncoding RNAs (ncRNAs) have a critical function in the control of this multi‐step pathway. Cdk9 is a serine‐threonine protein kinase that has been recognized as a regulator of cardiac differentiation. Recently, we have reported that. Cdk9 induces differentiation in the early stages of myogenesis, however its potential effects during differentiation is poorly understood. In this study, the regular mechanism of Cdk9 was investigated on the MRFs and myomiRs during myoblast differentiation. We continuously overexpressed Cdk9 during differentiation and quantified the expression of MRFs including MyoD, Mef2, and Myogenin. In addition, the transcription level of myomiRs consist of miR 1, 133 and 206 was examined upon Cdk9 overexpression during C2C12 myoblast differentiation.
Methods: We over-expressed Cdk9 in mouse myoblast C2C12 cells to investigate its regulatory roles on myogenic differentiation. Upon Cdk9 over-expression, the expression level of myogenic regulatory factors was determined. Moreover, the expression profile of three important myomiRs consist of miR 1, 133 and 206 was examined during the differentiation process via quantitative real‐time reverse transcription PCR (RT‐qPCR) and Western blot data analysis. Immunocytochemistry (ICC) was carried out following the manufacturerʼs instructions. In addition, Cell viability and apoptosis rate were determined via‐Annexin V FLUOS Staining Kit.
Results: The expression level of the most important MRFs including MyoD, Mef‐2, and Myogenin was determined in transfected cells during differentiation. Initially, the expression level of all three myogenic factors was increased in transfected cells compared with control cells, indicating the effects of Cdk9 overexpression during differentiation. Apart from MyoD transcription level which decreased in transfected and control cells, Mef2 and Myogenin expression level induced in control cells whereas, they were inhibited during differentiation in transfected cells. The expression of miR‐1/206 was increased while miR‐133 decreased in transfected cells in comparison with control groups. Moreover, the expression level of myomiRs was detected in transfected cells at days 1, 3, and 5 of differentiation. Our results showed that, although Cdk9 expression is necessary for inducing differentiation in the early stage of myogenesis, continuous Cdk9 expression inhibits differentiation by modulating myomiRs and myogenic gene expression.
Conclusion: These findings showed that, a transient induction of Cdk9 expression during myogenesis in the control cells in which the expression was induced in the early stage and declined toward terminal differentiation. Furthermore, in the previous study we have shown that Cdk9 transient induction by the destruction of a locus specific antisense transcript results in an efficient cardiac differentiation. These findings showed that Cdk9 likely modulates the expression of MRFs including MyoD and Mef2 as well as myomiRs, especially miR‐1 and miR‐133, in the early stage of myogenesis. In overall, the induction of myomiRs and transgenic factors relies straightly on Cdk9 or might be secondary to the complex regulatory network between transgenic genes and cardiac‐specific microRNAs. Finally, our results indicate that the transient induction of Cdk9 in the early stage of differentiation is critical for myogenesis.