| The heart of vertebrate is the earliest organs of form and function during embryonic development, and mainly derived from mesoderm mesenchymal cells, which occurred through multiple cell lines specialization, tubular structure formation, finally precise assembly into 4 heart cavity structure development process, need a series of important form events, including cell determination, migration and differentiation. The spatio-temporal order of these processes suggests there is a complex gene regulation pattern to ensure coordinated development of the embryonic heart. In recent years, the heart development research has made great progress, but the molecular mechanism of heart disease is still not very clear, more molecular mechanism research need further study. The emergence of microRNA, bring us a new perspective.microRNAs are a large class of evolutionarily conserved, small, noncoding RNAs, typically 22 nucleotides (nt) in length, that primarily function post-transcriptionally by interacting with the 3' untranslated region (UTR) of specific target mRNAs in a sequence-specific manner. In recent years, a large number of studies show that microRNA play an important role in the process of heart development, myocardial hypertrophy, heart failure, arrhythmia, hypertension, and so on, make the study of microRNA in the cardiovascular research fields become very popular all over the world.P19 cells are multipotent and can differentiate into cardiac myocytes in the particular conditions. Their gene expression are similar with mouse embryonic development. P19 cells are an excellent model system for the study of heart development, gene regulation. This study was to get the differential expression of microRNAs in the cardiac myocytes compared to undifferentiated P19 cell using microRNA chip technology and then select one microRNA to study its function. In the first part of this study, we investigated changes in microRNA expression profiles during differentiation of P19 cells into cardiac myocytes in order to elucidate the mechanisms of heart development. The morphology of P19 cells during differentiation was observed using an inverted microscope. Western blot analysis was performed to detect cardiac troponin I (cTnI) expression. Total RNA was extracted from P19 cells for microarray and real-time quantitative reverse transcription-polymerase chain reaction (real-time qRT-PCR) analyses to determine the microRNA expression profile. The microRNA microarray revealed differential expression of 49 microRNAs, of which 26 were down-regulated and 23 were up-regulated in differentiated cardiac myocytes, compared to normal P19 cells. This was confirmed by real-time qRT-PCR. We also utilized target prediction analysis to identify gene targets. We thus obtained the mmu-mir-19b target gene wnt1. In the second part of this study we selected microRNA-19b to study its function. we examined the effect of microRNA-19b on P19 cells differentiation,proliferation and apoptosis in vitro by establishing a stable cell line overexpressing microRNA-19b. We found that: (1) the morphology of P19 cells during differentiation and cardiac troponin I (cTnI) expression indicated that microRNA-19b inhibites differentiation of P19 cells. (2) the result of the CCK-8 assay indicated that microRNA-19b promote proliferation of P19 cells. (3) microRNA-19b can inhibites apoptosis induced by serum deprivation in P19 cells with the analysis of annexin V-FITC and Hoechst 33342 staining.In conclusion, we investigated changes in microRNA expression profiles during the differentiation of P19 cells into cardiac myocytes. We also obtained the microRNA-19b target gene wnt1 utilizing target prediction analysis. Finally we examined the effect of microRNA-19b on P19 cells differentiation, proliferation and apoptosis in vitro by establishing a stable cell line overexpressing microRNA-19b. It will provide new clues and theoretical basis for the prevention and therapy of congenital heart disease. |
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