| BackgroundCongenital heart diseases(CHD)are the most common kind of birth defect and include a series of cardiovascular malformations.It is believed that it is the result of genetic factors and environmental factors.Cardiac development is a delicate process and gene expression related to heart development is strictly regulated.In addition to the alteration of gene expression level caused by gene sequence,more and more attention has been paid to the important role of environmental factors in gene expression through epigenetic mechanisms.The epigenetic mechanisms include methylation modification,micro RNA regulation and histone modification.Currently,methylation is the most studied epigenetic phenomenon.DNA methylation is one of the most common epigenetic modifications.It mainly refers to the transfer of methyl S-adenosylmethionine to the 5'-position of DNA cytosine by DNA methyltransferase to form 5-methylcytosine.DNA modification does not alter gene sequences,but it can regulate gene expression,maintain chromosomal integrity,regulate DNA recombination and transcriptional activity of some genes.Promoter is the DNA sequence located upstream of 5'-terminal of structural gene.It can activate RNA polymerases to bind to DNA template accurately and control the initiation and level of gene expression(transcription).The regulation of gene transcription by promoters is mainly manifested by the cooperation among cis-acting elements,trans-acting factors and RNA polymerase.Its essence is the interaction between protein and DNA,protein and protein.Variations in the promoter sequence can lead the changes of gene transcription levels by affecting the binding of trans-acting factors to cis-acting elements.In addition to sequence changes,more and more studies have shown that changes in DNA methylation levels in gene promoter regions can regulate transcriptional levels.The methylation of CpG island in promoter region will affect gene transcription and inhibit its expression and function.While demethylation can induce gene reactivation and expression.Methylation and demethylation control gene expression mainly through direct and indirect mechanisms.The direct mechanism is that CpG island methylation can prevent the gene expression directly by inhibiting RNA polymerase activity or interfering with the binding of transcription factors to gene regulatory regions.The indirect mechanism mainly consists of t.^wo types:1.The methylation of the promoter regulatory sequence binds to the methylated CpG binding protein and prevent the formation of transcription complexes between genes and transcription factors;2.The DNA methylation in the promoter changes the chromatin structure and blocks the binding of transcription factors to DNA.There are mainly four types of molecules involved in heart development:transcription factors,signaling molecules,cell adhesion molecules and ion channel molecules.Transcription factors are the most studied,including zinc finger family,homologous domain family,T-box family,basic helix-ring-helix and MADS domain family.Transcription factors are proteins that bind to specific sequences of nucleic acids and regulate gene expression by recognizing and binding cis-acting elements in the promoter of genes.Transcription factors act on downstream genes and are also regulated by upstream-induced signals.They form a complex and accurate network and play important roles during heart development.T-box family belongs to the family of transcription factors related to developmental regulation.It participates in the development of many organisms through its unique T-BOX region.T-BOX is a large DNA binding domain and all the DNA sequences that it binds to have a consistent sequence TCACACCT(T-half site).The genes encoding the T-BOX protein are called T-box genes.T-box gene family is divided into five different subfamilies.TBX1,TBX18,TBX20 in the TBX1 subfamily and TBX2,TBX3,TBX5 in the TBX2 subfamily are associated with heart development.They play an important role in the development of the heart through synergistic and competitive mechanisms.Tetralogy of Fallot(TOF)is the most common cyanotic congenital heart disease,accounting for about 10%of congenital heart disease.It is caused by abnormal development of the conical trunk during embryonic period.Pulmonary stenosis,ventricular septal defect,aortic straddling and right ventricular hypertrophy are the main pathological features.The natural prognosis of TOF patients is poor.Most of them die of secondary cardiac hypertrophy and heart failure caused by chronic hypoxia before adulthood if they are not diagnosed and treated in time.A small number of TOF patients have sudden death due to postoperative arrhythmia and other complications.TOF is the most common clinical malformation of the trunk of conus arteriosus.Some patients have chromosome and gene mutations.No clear pathogenic gene is reported yet.TBX20,TBX5,TBX1,Cx43,NKX2.5,GATA4,FOG2,HIRA,TGF beta 2,VANGL2 and so on are thought to be related to TOF.TBX20(T-box20)locates on chromosome 7p14.2 and belongs to TBX1 subfamily.The study of mouse embryos showed that TBX20 had strong expression in the endocardial cushion at the early stage of cardiac tube formation.It was more significant in the outflow tract,atrioventricular canal and atrial septum anterior border of endocardial cushion in the later cardiac development.TBX20 is the only member of the T-box family that can express in the endocardial cushion stably and widely.The abnormal development of endocardial cushion can lead to a variety of congenital heart diseases,including transposition of great artery,double outlet of right ventricle,TOF,atrioventricular septal defect and so on.Therefore,TBX20 is one of the candidate genes for conus arteriosus malformation in human.TBX2 gene is located on chromosome 17q23,which is widely expressed in various tissues and organs in the embryonic development.Abnormal expression of TBX2 in mice is often accompanied by abnormal expression of specific genes in outflow tract septal defect and atrioventricular canal.Previous studies have shown that TBX20 can promote the expression of specific genes in chamber myocardium and inhibit the expression of TBX2 which plays an important role in the proliferation and differentiation of cardiac myocardium.Cai et al reported that BMP2 was one of the key downstream genes of TBX20 in the development of atrioventricular canal.They believed that TBX20 not only directly inhibited the expression of TBX2,but also indirectly promoted the expression of TBX2 through BMP2 signaling pathway.Previous studies have shown that TBX20 mutation occurs only in a small number of CHD patients.The alterations in the TBX20 sequence do not fully explain its pathogenic role in CHD.At present,the gene methylation research in human is mainly concentrated in tumor.The role of DNA methylation in CHD is little studied.Objectives1.To detect the methylation level of TBX20 gene promoter and the expression levels of TBX20 mRNA and protein in the right ventricular outflow tract myocardium of TOF patients.To explore the relationship between the methylation level of TBX20 promoter and the expression of TBX20.2.To analyze the mRNA and protein expression levels of TBX20 related factor TBX2 and BMP2 in the myocardium of TOF patients.Explore the relationship between them and reveal their pathogenicity functions in TOF.3.Explore the relationship between the methylation of TBX20 promoter and TOF.To supply a model for further study of the pathogenesis of TOF and to provide a theoretical basis for perinatal screening of congenital heart disease.Methods1.Using National Center for Biotechnology Information(NCBI)site to find the TBX20 promoter sequence of upstream of 2000bp and downstream of1000bp.Methyl Primer Express Software v1.0 was adopted to find the distribution of CpG island in promoter of TBX20.Neural Network Promoter Prediction was adopted to examine the core promoter sequence of TBX20 gene.JASPAR was adopted to predict the binding sites of TBX20 CpG island with other transcription factors.2.Myocardium tissue of right ventricular outflow tract in 23 children with TOF and 5 children who died from accident were selected as subjects.Methylation of TBX20 gene promoter region was detected by bisulfite PCR sequencing.The mRNA and protein expression levels of TBX20 and its related factors TBX2 and BMP2 were tested by fluorescence quantitative PCR and western blotting respectively.3.Statistical analysis:All data were analyzed on SPSS 22.0 and GraphPad prism software v5.Normal distribution data were represented by mean(±)standard deviation,and non-normal distribution data were represented by median and quartile.The normal data of TBX20 promoter methylation level,relative expression of mRNA and protein were analyzed by t-test,and the non-normal data were analyzed by Mann-Whitney test.Spearman was used to test whether there was correlation between TBX20 and mRNA.P<0.05 was statistically significant.Results1.TBX20 promoter(-400bp?-48bp)is located on CpG island by bioinformatics website and software.There are 42 CpG loci in this region.It was predicted that there were multiple transcription factor binding sites(TFAP2,GATA5 and TBX1/2/4/5,etc.).2.The total methylation levels of TBX20 promoter region(-400bp?-48 bp)in TOF group were significantly lower than those in control group(P=0.035).In the 42 CpG sites,the methylation levels of 26th CpG site in TOF group were lower than those in control group(P=0.016).There was no significant difference in other CpG sites between the two groups.3.The mRNA expression levels of TBX20 in right ventricular outflow tract myocardium of TOF group was significantly higher than those of in the control group(P<0.001),the difference was statistically significant.The methylation level of TBX20 promoter region(-400bp to-48 bp)in the right ventricular outflow tract myocardium of TOF group was negatively correlated with the expression level of TBX20 mRNA(r=0.81,P<0.001).The expression of TBX20 protein in right ventricular outflow tract myocardium of TOF group was higher than that of control group(P =0.080).The difference was statistically significant.4.The mRNA expression level of TBX2 in right ventricular outflow tract myocardium of TOF group was lower than that of in the control group(P<0.001).There was no significant difference in BMP2 mRNA expression between TOF group and the control group(P=0.082).5.The expression of TBX2 protein in right ventricular outflow tract myocardium of TOF group was significantly lower than that of in the control group(P=0.022).There was no significant difference in BMP2 protein expression between TOF group and the control group(P=0.537).Conclusions1.There were multiple transcriptional regulatory elements in TBX20 promoter region(-400bp to-48bp).The decreased methylation of TBX20 promoter could affect the binding of TFAP2C and TBX20,which can increase the expression of TBX20 mRNA and protein.2.Increased expression of TBX20 can inhibit the expression of TBX2 mRNA and protein.Exceptional expression of TBX20 and TBX2 genes can both lead to the abnormal cardiac development,which may be one of the pathogenic mechanisms of TOF development.3.The methylation level of TBX20 gene promoter region may play an important role in the pathogenesis of TOF.This study provides a new research model for further exploring the pathogenesis of TBX20 gene in tetralogy of Fallot.Meanwhile,it supplys a new theoretical basis for perinatal screening of congenital heart disease. |
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