| Background:Congenital hypospadias is one of the most common congenital developmental abnormalities of the male genitourinary system.Epidemiological surveys had found the incidence of hypospadias had increased by three times in the past two decades.Hypospadias is main treated by the surgery,sometimes requiring multiple surgeries,especially in the severe hypospadias to achieve the result in satisfactory in function and appearance.Many patients with hypospadias had all kinds of the post-operation complications,such as urethral fistula and urethral stricture,resulting in lifelong difficulty in urination and sexual function,and increasing the risk of psychological problems,which brings serious physical,mental and economic burdens to individuals and families.Therefore,the pathogenesis and treatment of hypospadias have attracted more and more attention from researchers.At present,hypospadias is generally considered to be a multifactorial and multi-stage process involving a complex interaction of genetic and environmental factors.Epidemiological statistics have found that environmental factors are strongly associated with the occurrence of congenital hypospadias.However,only a small number of relevant exposed individuals eventually developed congenital hypospadias,indicating that individual genetic susceptibility is also involved in the development of hypospadias.In the context of the great achievements of the Human Genome Project,important advances have been made in genetic susceptibility studies of various common complex diseases marked by Single Nucleotide Polymorphism(SNP),including candidate gene association study and genome-wide association analysis Association Study(GWAS),association analysis has become one of the hot spots in the study of complex disease genetics in the post-genomic era.Therefore,using SNP as the starting point to explore the mechanism of hypospadias is expected to open up a new way for the early diagnosis of hypospadiasEmbryogenesis studies have found that there are three main molecular pathways that affect the formation of male external genitalia:androgen dependent,androgen-dependent and dependent on endocrine and environmental factors,and they also show complex interaction patterns,mutual influence,and ultimately promote the normal development of male external genitalia.Current research on hypospadias has focused on androgen-related pathways.The pathway of GATA4-SRY-Sox9 plays a decisive role in the process of male sex determination,and GATA4 gene is highly expressed in the developing gonads during sex development.At the same time,under the action of MAPK kinase signaling,GATA4 protein phosphorylation is activated,which works synergistically with NR5A1,FOG2 and other proteins to participate in regulating the expression of SRY,Sox9 and anti-Müllerian hormone gene(AMH).Therefore,abnormalities in the expression of genes at any stage involved in the formation of male genitalia can lead to malformations of the genitourinary system,including hypospadias.Therefore,based on the importance of the GATA4 gene during embryonic development,combined with the production process and mode of action of GATA4 gene.In our research,GATA4 was selected as the candidate gene,and the SNPs association analysis strategy of candidate genes were used to explore the association between GATA4 SNPs and the genetic susceptibility of hypospadias,and the functional studies of the positive SNP sites were investigated.This paper analyzed the potential molecular mechanism by which miRNA regulate the GATA4 expression to cause the pathogenesis of hypospadias,explored the possible mechanisms of the GATA4-SRY-Sox9 pathway and hypospadias and provided a reliable theoretical basis for the prevention and treatment of hypospadias.Part Ⅰ.Association of GATA4 gene polymorphisms with susceptibility to hypospadias in Han Chinese childrenObjective:We investigated that the relationship between GATA4 polymorphisms and susceptibility of hypospadias,statically analyzed the association between different genotypes and the pathogenesis and progression of hypospadias and validated the differences between different genotypes of positive SNP sites for GATA4 in vivo.Methods:1.We genotyped four potential functional polymorphisms(rs128458,rs12825,rs884662,and rs904018)in GATA4 by the TaqMan MGB method in a hospital-based two-phase casecontrol study involving a total of 410 children with hypospadias and 520 normal control children.2.Univariate and multivariate logistic regression was used to assess the association of different genotypes with the risk of hypospadias,and adjusted for potential confounding factors.3.Chi-square tests were used to assess differences in demographic variables between cases and controls and distribution of GATA4 polymorphisms between subgroups.4.Stratified analysis was used to assess the difference between positive loci in different clinical types of hypospadias and the corresponding diseases.5.RT-PCR was used to detect the difference in expression levels of GATA4 mRNA in different genotypes of rs128458A>T at the positive S NP locus in normal tissues of hypospadias.Results:1.We found there was no statistically vital difference in the frequency of distribution among maternal gestational age during pregnancy,maternal previous birth history,pregnancy cycle at delivery,folic acid supplementation in our case-control studies,estrogen supplementation during maternal pregnancy and low birth weight infants were risk factors for hypospadias.2.In a two-phase case-control study,A significant association was found between rs12458(3’-UTR of GATA4)and susceptibility to hypospadias(P=0.008).Compared with rs12458 AA genotype individuals,those harboring the variant allele(rs12458 AT/TT)were correlated with significantly higher risk of hypospadias(AT/TT vs AA,OR=1.42,95%CI=1.17-2.35,P=0.036).And rs 12825 C>G was associated with the incidence of hypospadias in the first-stage study,but there was no statistically significant correlation after expanding the sample size.3.A stratified analysis of cases in children with hypospadias found that children with the rs12458 T allele and those with the GG/GC genotype with rs904018 may have an increased risk of congenital heart disease and cryptorchidism,independent of hypospadias severity.4.The expression levels of GATA4 mRNA carrying TT genotype and AT/TT genotype were significantly lower than those of the AA genotype group in hypospadias.Conclusions:1.Maternal estrogen supplementation during pregnancy and low birth weight infants are risk factors for hypospadias.2.The present results indicate that the functional GATA4 rs 12458 variant confers individuals’susceptibility to hypospadias,children with the T allele have a significantly increased risk of hypospadias.3.GATA4 mRNA carrying a mutant genotype(T allele)is expressed at lower levels than wildtype genotypes(A allele)in the hypospadias tissue.Part Ⅱ.A preliminary study of the functional nature of the GATA4 rs12458 A>T gene polymorphismObjective:We investigated the biological functional properties GATA4 rs12458A>T in hypospadias and explored the lying mechanisms of GATA4-SRY-Sox9 axis in the pathogenesis and progression of hypospadias in vitro.Methods:1.We co-transfected Luciferase reporter plasmids containing the GATA4 rs 12458 wild-type(GATA4-rs12458A)and Mutant(GATA4-rs12458T)Alleles in the HEK-293T cell line.2.We used the Real time RT-PCR and the Western Blot to test the mRNA and protein levels of GATA4 and the expression of its downstream genes SRY and Sox9.3.We tested the cell proliferation by the CCK-8,the migration ability of cells by the Wound Healing and the cell Apoptosis level by the flow cytometry.4.The wild-type and mutant-type of GATA4 rs12458 were cloned into dual-luciferase reporter vector and overexpression vector respectively,and the dual-luciferase reporter vector and hsa-miR-556-5p mimic were co-transfected into cells to detect the expression of luciferase.5.We detected changes in GATA4 mRNA and protein levels after co-transfection of plasmids and hsa-mir-556-5p into cells by RT-PCR and Western Blot.Results:1.We found that the mRNA and protein expression levels of GATA4,SRY and Sox9 were significantly decreased in the mutant T allele compared with the wild type A allele,the difference was statistically meaningful(P<0.05).2.Mutant T allele of the Gata4 significantly inhibited the proliferation and migration in HEK293-T Cells compared with the control group(P<0.05).The results of Flow cytometry test showed that the mutant T allele Gata4 could promote the apoptosis of HEK293-T cells,but there was no significant difference(P>0.05).3.We predicted the mutation of rs12458 might have created a binding site for hsa-mir-556-5p in the 3’-UTR region of GATA4 through the bioinformatics analysis.Furthermore,the luciferase reporter assays revealed the rs 12458 mutant T allele showed significantly decreased activity compared with the wild type A allele(P>0.05).4.Under the intervention of hsa-mir-556-5p,the expression of GATA4 mRNA and protein in the mutant T allele group was significantly lower than that in the wild type A allele group(P<0.05),indicating a possible role of rs 12458 variant in regulating the combination of microRNAs with the GATA4 mRNA.Conclusions:1.GATA4 rs12458A>T can lead to a decrease in the expression levels of GATA4,SRY,Sox9 mRNA and protein in cells,and inhibit the proliferation and migration ability of cells2.The mutant T allele of GATA4 rs12458,down-regulated the GATA4 expression by increasing the binding site of hsa-mir-556-5p.3.The interaction between hsa-mir-556-5p and GATA4 rs12458 A>T polymorphism might inhibit cell proliferation and migration by regulating the expression of SRY and Sxo9 which are the important genes in reproductive development,result in hypospadias. |
