The Influence Of MiR-145 Over Expression Impacts On Zebrafish Early Heart Development And The Primary Studying On Its Mechanism

MicroRNAs comprise a novel class of endogenous, small(only about 20 to 25 nucleotides), and noncoding RNAs, and almost involved in the physiologic and pathologic process of cardiovascular system. MicroRNAs, in common, play the similar roles as transcription factors by specifically binding the seed sequence within 3'UTR of target genes, by activating their degradation or inhibiting their translation of the target genes mRNAs or inhibit their translation, which result in variety of cell activities changing at different levels. Moreover, during the each stage of development, microRNAs maintain the shift balance between differentiation and proliferation of cell-status. MiR-145 mature sequence is highly conserved between different species. Seed sequences are also highly conserved by comparing their orthologs 3'UTR among different species.The heart is the first functional organ to develop in vertebrate embryos. The early development of zebrafish heart consists of four important stages. Firstly, cardiac progenitors existing in the lateral marginal zone on either side of the embryo differentiate and migrate towards the midline into the anterior lateral plate mesoderm. Secondly, the progenitor cells further migrate to form the heatr tube. Later, the heart tube begins to constrict at the future AV canal and loop. Finally, after a series of complex morphological changes, including obvious atrium and ventricle appear, heart develops into its mature shape. Heart plays its important functions due to the cardiac contraction and diastolic normally. As the essential components of myofibrils in cardiomyocytes, sacomeric proteins, including myosin heavy chain and light chain, regulate the cardiac contraction and diastolic fundamentally.Myosin light chain is composed of the essential and regulatory units, named as ELCs and RLCs. Mutations of ELCs and RLCs in human might lead to hypertrophic cardiomyopathy(HCM). Cmlc1 and cmlc2 are identified as the main ELC and RLC orthologues in zebrafish. In this study, we focus on cmlc1 and cmlc2. The bioinfomational analysis shows that miR-145 potentially targets cmlc1 3' UTR. We found that over-expression of mi R-145 may cause abnormal phenotypes of zebrafish hearts during the early development. We also found that miR-145 could specifically binds to cmlc1 3 'UTR, and inhibits the genes expression in upstream through the methods of constructing PCS2-EGFP-cmlc1 3 'UTR plasmid and its report assay. Finally, we detected the RNA expression changes of cmlc1 and cmlc2 caused by miR-145 over-expression. Our results suggested that one of the most important mechanisms of miR-145 affecting the cardiac function might be due to its binding to cmlc1 3' UTR seed sequence, and then resulting in the morphological structure and physiological function of zebrafish heart during its early developmental process.