DNA Methylation Analysis Of The SOCS3Promotor And The Possible Mechanism In The Myocardium Of Infants With Cyanotic Congenital Heart Defects

BackgroundCyanotic congenital heart defects threaten human life and health, and theunderstanding of the pathophysiological mechanism facilitates clinical diagnosis, treatmentand prognosis. Such patients undergo the common pathophysiological process, chronichypoxia. However, it is not completely clear how the myocardium faced with chronichypoxia stress achieve the adaptive transformation of structure and function includingapoptosis, autophagy, secretion, metabolism, remodeling.Suppressor of cell signaling-3(SOCS3), a downstream signal molecule of Janus kinase-Signal transducer and activator of transcription-3(JAK-STAT3), negatively regulating the s-ignaling pathway, maintains the balance of the cell signaling pathway. Our previous studyfound that the activation JAK-STAT3, and high level of SOCS3mRNA and protein in themyocardium of patients with cyanotic congenital heart defects compared to acyanotic ones, and the protein level of SOCS3was dependent of transcription level. Moreover, hypoxia cardiomyocytes overexpressing SOCS3inhibited IL-6-JAK-STAT3activation and cardiomy-ocytes apoptosis increased. It suggested moderate expression of SOCS3, which was pivotalfor IL-6-JAK-STAT3signaling balance, played a important role in the regulation ofmyoc-ardial adaption to chronic hypoxia. However, the mechanism that regulated the expression of SOCS3was not clear yet. The tumor research revealed that，methylation of SOCS3promotor played a role in the transcription, and affected protein expression of SOCS3, accordingly regulating JAK-STAT3activity，maintaining tumor cell proliferation and anti-apo-ptosis. Thus, we speculate that in the chronic hypoxic myocardium, hypomethylation ofSOCS3promotor might play a role in the regulation of transcription, modulating JAK-STAT3signaling pathway in the myocardial adaption to chronic hypoxia. Our study wou- nd research the molecular mechanism of myocardial adaption to chronic hypoxia fromthe perspective of epigenetics, in order to establish a theoretical basis of the molec-ular target of myocardial protection in the perioperative period.ObjectivesThe purpose of this study was to investigate the methylation level of Suppressor of cellsignaling-3(SOCS3) promotor and the possible mechanism in the myocardium of infantswith cyanotic congenital heart defects.Methods1.Collect right ventricular myocardium samples of infants with cyanotic (cyanoticgroup,n=18) and acyanotic congenital cardiac defects (acyanotic group,n=16). Extractgenome DNA from myocardial tissue, and detect the methylation level of SOCS3promtorCpG island with Methylation specific PCR(MSP) and Bisulfite Sequencing PCR(BSP).2.Protein level of DNA methyltransferase1(DNMT1),DNA methyltransferase3A(DNMT3A), DNA methyltransferase3B(DNMT3B) were examined by Western blotting.Transcription level of DNMT1, DNMT3A, DNMT3B were detected by quantitativeReal-time polymerase chain reaction (qRT-PCR).Results1. We observed that the partial methylation of SOCS3promotor in the cyanoticgroup(Methylation level=54.287±16.744%), and non-methylation of SOCS3promotor inthe acyanotic group(Methylation level=0.000±0.000%).2. Compared to acyanotic group, cyanotic group displayed higher protein level ofDNMT3A(0.407±0.469v.s0.160±0.034, P<0.05), similar protein level of DNMT1(0.084±0.115v.s0.081±0.085, P>0.05) and DNMT3B(0.054±0.012v.s0.052±0.093, P>0.05).3. Compared to acyanotic group, cyanotic group showed obvious decreased expressionof the mRNA of DNMT1(0.548±0.553v.s0.920±0.456, P<0.05), DNMT3A(0.555±0.395v.s0.756±0.240,P<0.05) and DNMT3B(0.473±0.509v.s1.018±0.749,P<0.05).4. Cyanotic group showed a significant correlation between DNMT3A protein leveland the methylation level of SOCS3promotor(r=0.359，P=0.047).ConclusionsIt showed that the partial methylation of SOCS3promtor CpG island and elevatedlevel of DNMT3A protein in the myocardium of infants with cyanotic congenital heart defects, and there was a significant correlation between DNMT3A protein level and themethylation level of SOCS3promotor. DNMT3A might establish and maintain themethylation of SOCS3promtor CpG island through enzymatic reaction, being recruited andanchoring to nucleosomes containing methylated CpGs.