The Study Of The Relationship Between Nodal Signaling Pathway Gene NOMO1 And Embryonic Heart Development

Nodal Modulator 1(NOMO1) gene is one of three highly similar genes in a region of duplication located on the p arm of chromosome 16, which encodes a 1222-amino acid protein. Its molecular weight is about 130 KD. NOMO1 and its binding parter Nicalin, as part of a endoplasmic reticulum-located membrane protein complex, are novel Nodal signaling antagonists. Nodal signaling pathway is critical for the formation of the heart and other visceral organs.In a previous study using suppression subtractive hybridization (SSH), we identified 551 genes that were differentially expressed in ventricular septal defect (VSD) and normal ventricular septum myocardium. We found that NOMO1 gene was downregulated in VSD myocardium, and might play a significant role in the development of human congenital heart disease (CHD).In this study, we aimed to investigate the change of NOMO1 gene mRNA expression and its loctation during development of rat embryonic hearts. Subsequently, we took advantage of murine embryonic carcinoma cells(P19 cells), which could undergo myocardial cell differentiation after treatment with 1% dimethylsulfoxide (DMSO), to investigate the effects of NOMO1 gene silencing by RNA interference (RNAi) on cardiomycytes differentiation. Further study of the function of NOMO1 and the analysis of the relationship between NOMO1 and cardiogenesis, may help to clarify the etiology of CHD and aid in improving diagnosis as well as in developing novel therapeutic strategies, and to explore its significace in embryonic heart development.Part ⅠExpression of nodal modulator 1(NOMO1) gene during development of rat embryonic heartsObjective:To investigate the change of NOMO1 gene mRNA expression and its loctation during development of rat embryonic hearts, and to explore its significance in embryonic heart development.Methods:24 pregnant Sprague-Dawley rats were randomly divided into 6 groups(n=4) and embryonic hearts were obtained on the 12th、15th、16th、17th、 19th、21th in pregancy. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) was used to reveal NOMO1 gene mRNA expression, fluorescence in situ hybridization (FISH) for its positioning analysis.Results:With the development of rat embryonic hearts, the expression levels of NOMO1 gene mRNA were from weak to strong signs of abating and its peak was on 16th day. NOMO1 gene was mainly expressed in the epicardium, while it was not detected in the myocardium, endocardium and heart valves.Conclusion:The expression of NOMO1 gene is dynamic, and it may play an important role in the fetal heart development.Part IISilencing of NOMO1 gene inhibits the differentiation of P19 cells into cardiomyocytesObjective:To investigate the effects of NOMO1 gene silencing on the differentiation of P19 cells into cardiac myocytes, and to explore its significance in embryonic heart development.Methods:①We constructed murine NOMO1 pGPU6/GFP/Neo-shRNA interference vector to silence NOMO1. These vectors, as well as empty ones were transfected into P19 cells. Stable NOMO1 silenced cells were selected using G418 and their differentiation into cardiac myocytes was induced using 1% DMSO. Subsequently, real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) and Western Blot analysis were used to verify NOMO1 gene mRNA and protein expression levels on days 0、6、10 of differentiation. ②The morphology of P19 cells during differentiation was observed under inverted phase contrast microscope.We used real-time qRT-PCR and Western Blot techniques to detect Nodal signaling pathway related genes Nodal、Cripto and Smad2 genes mRNA and protein expression levels on days 0、6、10 of differentiation. We solely used real-time qRT-PCR analysis to detect myocardial marker gene CTnT and transcriptional factor Nkx2.5、GATA4、TBX5 mRNA expression levels.Results:①In RNA interference group (RNAi), NOMO1 gene mRNA and protein expression levels were significantly lower than the negative control group (NC), which achieved a good efficiency of RNA interference. ②The cardiac myocardial cells aggregated on day 5 of differentiation showed similar morphological changes between NC group and RNAi group. On day 10 of differentiation, beating cells began to appear, which were hardly observed in RNAi group. Nodal signaling pathway related to genes Nodal, Cripto and Smad2 mRNA and protein expression levels significantly decreased in RNAi group compared with NC group, and the trends of these two groups expressive chang were similar. CTnT、NKX2.5 mRNA levels in RNAi group increased slightly on day 6 compared with NC group, while on day 0 and day 10, the levels significantly decreased. GATA4 mRNA levels in RNAi group significantly decreased compared with NC group. TBX5 mRNA levels in RNAi group increased slightly on day 0 compared with NC group, while on day 6 and day 10, the levels significantly decreased.Conclusion:Silencing of NOMO1 gene inhibits the differentiation of P19 cells into cardiomyocytes, and it may play an important role in the fetal heart development.