Relationship Of Mutation And Methylation In CITED2with Congenital Heart Disease

Objective：Explore CITED2mutation that how to impact the expression of thetranscription factor HIF-1α, and to determin the role of methylation ofCITED2promoter in congenital heart disease, so that help us to understandthe pathological mechanisms of congenital heart disease.Methods:CITED2(c.573-578del6) mutant recombinant plasmid and wildrecombinant plasmid were successfully constructed in our early study.Transfecting them into HepG2and H9C2respectively. The cells werecultured in hypoxia. RNA was extracted and transcripted into cDNA after24h, Expression differences of HIF-1α mRNA were compared amonggroups by Q-PCR.And the expression of HIF-1α protein were detected byWestern-blotting after48h. Myocardial tissues from31children withcongenital heart disease was collected during surgery in department ofcardiothoracic surgery from March2011to March2012,and tissuessamples of2health children were harvested after accidental death.Bisulfite-sequencing PCR (BSP) and methylation-specific PCR(MSP) were used to detect methylation in CITED2promoter. And thenexpression differences of CITED2mRNA were compared between themethylate and control group.Results:1. Expression of HIF-1α mRNA was down in wild group comparingwith the empty vector group(P<0.05),but it was higher in mutant groupthan that of wild group(P<0.05),empty vector group and no transcriptiongroup showed no significant difference.2. Expression of HIF-1α protein was down in wild group comparingwith the empty vector group(P<0.05),but it was higher in mutant groupthan that of wild group(P<0.05),empty vector group and no transcriptiongroup showed no significant difference.3. CITED2methylation was found in10of12congenital heart diseaseby BSP, positive rate of CITED2methylation was83.3%(10/12).4. CITED2methylation was found in16of19congenital heart diseaseby MSP, positive rate of CITED2methylation was84.2%(16/19).5. Abnomal methylation was not found in the control group.6. Compared with the control group,CITED2mRNA expression wassignificantly reduced(P<0.05).Conclusion:1. HIF-1α can be negative feedback inhibited by CITED2. 2. The negative feedback inhibition was breaked by CITED2mutation.3. The CITED2mutation leads to overexpression of HIF-1α,whichmaybe one of factors of the incidence of congenital heart disease.4. Abnomal methylation of CITED2promoter (CpG island) is found incongenital heart disease.and the abnomal methylation leads to decreasemRNA expression of CITED2.5The abnomal methylation of CITED2might be involved in theincidence and development of congenital heart disease.