The Important Role Of Rbm24and Preliminary Screening Of Related Signaling Molecules In Cardiac Cells

Objective:This project aims to investigate the molecular and biochemical mechanisms of Rbm24in cardiac differentiation and cardac myocytes. Through the way to find the target mRNA of Rbm24and its binding proteins, we can reveal the role of Rbm24in stem cells differentiation. Then we can reveal Rbm24knockdown related signaling pathways resulting cardiomyopathy and further to provide a research basis and an important novel target for the treatment of cardiomyopathy.Method:qRT-PCR was used to detect the expression of Rbm24protein in the process of ESC differentiation. Meanwhile, we use Western blot and immunofluorescence staining to detect the expression of Rbm24protein in primary cardiomyocytes, H9C2and C2C12cell line. To analyze the impact of Rbm24knockdown on the expression of cardiac structural proteins and cardiac contraction, RNA interference technology are applied to build Rbm24knockdown cells siRNA-Rbm24. A stable transfection Rbm24-Flag overexpressing oocytes H9C2-23cell line was build by gene cloning and lipofectamine transfection technique. RIP-CHIP technology was adopted to provide systematic analysis of target mRNA of Rbm24and the results were verified with RIP-PCR. Meanwhile, CO-IP technology and mass spectrometry were adopted to provide systematic analysis of binding proteins of Rbm24and the results were verified with gene cloning and CO-IP.Results:In the third day of ESC differentiated into cardiomyocytes, Rbm24was barely detectable and cardiomyocytes differentiation starts. The expression of Rbm24increases by day6. Rbm24showed positive expression in a-actinin-labeled primary cardiomyocytes and evenly distributed in the nucleus and cytoplasm. Also, Rbm24uniformly distributed in the nucleus and cytoplasm in H9C2cell line and its expression was up-regulated specifically the cytoplasmic protein after the induction of differentiation. Rbm24didn’t express in C2C12myogenic cell line, and its expression was gradually increased after the induction of differentiation into myoblast which use the horse serum. These data suggested that Rbm24is an early marker of cardiac differentiation and a heart-enriched gene. On the one hand, knockdown of Rbm24reduces expression of sarcomeric proteins such as TNNT2, TPM, MYH6(α-MHC) and ACTN2. On the other hand, it leads to a pattern of intracellular filaments became less clear and Spontaneous contraction was reduced. These data indicate the important role of Rbm24in maintaining cardiac function. RIP-CHIP technology obtained the target mRNA of Rbm24and the results were verified that MYOG and Stat3bind with Rbm24. CO-IP combined with mass spectrometry obtained the target binding proteins of Rbm24, and further verifed through gene cloning and CO-IP.Conclusion:Rbm24protein is expressed in the process of ESC differentiation into cardiomyocytes and cardiac tissue. The data suggested that Rbm24is an early marker of cardiac differentiation and have an important role in cardiac cells and maintenance of myocardial contractility, which is likely to be used as an indication of clinical cardiomyopathy. Rbm24rugulated cardiac differentiation and the function of cadiac cells through its target mRNA and the proteins interacted.