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Background Conotrumcal defects (CTD), induced by abnormal development of the outflow tract (OFT), account for almost 30% of congenital heart disease (CHD) and can be categorized as TGA, DORV, tetralogy of Fallot, and PTA.It has been clearly clarified that RA (retinoid acid) signaling is essential for cardiogenesis including the development of OFT.As an important component of RA signaling pathway, the retinoid x receptor alpha (RXRA) is considered to play important role in pathogenesis of CTD.Indeed, deficiency of RXRA or germline mutation of this receptor in mice results in conotruncal defects and ventricular abnormality.Taken together, RXRA may be a potential candidate gene of tetralogy of Fallot (TOF) which involves ventricular defects and conotruncal abnormalities.Objective The aim of the study was first to investigate the expression level of RXRA in the right ventricular outflow tract (RVOT) myocardium tissue of TOF patients compared with controls and then to preliminarily clarify the possible regulation mechanism of RXRA in TOF patients.Methods The expression of RXRA mRNA in TOF patients and controls was detected by real-time PCR.To clarify the cause of the expression difference of RXRA, methylation status of the RXRA promoter region in TOF patients and controls were detected by BSP cloning-based sequencing.Dual-luciferase assay combined with methylation assay in vitro were performed to determine the transcription activity of unmethylated and methylated CpG region in RXRA promoter.Results Real-time PCR analysis revealed that the mRNA expression of RXRA in the RVOT myocardium was significantly decreased in TOF patients compared with the controls (p<0.0001).The methylation status of CpG region containing CpG sites 1~23 (-1000~-1453, relative to the TSS) in the RXRA promoter region was statistically higher in TOF patients than the controls.By searching the web site http://www.cbrc.jp/research/db/TFSEARCH.htrnl, we found that this CpG region contained several transcription factor sites including SP1.In addition, dual-luciferase assays combined with methylation assay in vitro showed that this CpG region had transcriptional regulate activity and methylation of this region can depress its transcriptional regulate activity.Conclusion To our knowledge, this is the first report that there is down-regulation of RXRA mRNA expression and aberrant promoter methylation status in the RVOT myocardium of TOF patients compared with the controls.The aberrant methylation at RXRA promoter may be responsible for the aberrant gene expression in RVOT myocardium of TOF patients.These findings suggest that TOF may also be an epigenetic disease.