MiRNAs Contribute To Congenital Heart Disease By Targeting TBX5 And Mediating Its Differential Expression Due To A Genetic Variant

Congenital heart disease (CHD) is one of the most common birth defects and the leading cause of infant morbidity worldwide. Considering the importance of early diagnosis and treatment, it is of great significance to understand the cause of CHD. Transcript factors are well known to maintain normal development of embryonic heart development. In human CHD studies, a mutation in coding region of TBX5 was the first identified variant, and it was thought as causative factor of Holt-Oram syndrome phenotype including atrial septal defect (ASD), ventricular septal defect (VSD), and conduction system abnormalities. In view of aberrant gene function due to amino acid changes, genetic variants in coding region of TBX5 have been extensively explored, while the variants in non-coding region,3’untranslated region (3’UTR) in particular, which can regulate gene dosage, have been ignored to a large extent.To study the association between genetic variants of TBX5 3’UTR and the susceptibility of CHD, we recruited 1,177 CHD cases and 990 healthy controls from Shanghai and Shandong. Among them,32 subjects were selected to undergo TBX5 3’UTR DNA sequencing. According to our screening results, four variants with minor allele frequency over 0.05 were chosen for genotyping by SNaPshot and association analysis both in Shanghai and Shandong group. Our case-control study in the two provinces both indicated that TBX5 3’UTR rs6489956 C>T might increase CHD susceptibility with T allele as the risk factor, i.e., the risk of CHD in CT-TT genotype was 1.9 times that of CC genotype (adjusted OR= 1.90,95%Cl= 1.52-2.38, P= 8.79×10-9). And in stratified analysis, this association was further confirmed in various CHD phenotypes, particularly in ventricular septal defect (VSD) (CT:OR= 1.61,95%CI = 1.23-2.10; TT:OR=2.43,95%CI=1.04-5.65, P=4×10-4).Following the discovery of the risk factor rs6489956, we then conducted functional analysis of the genetic variant. Quantitative reverse transcription PCR (qRT-PCR) showed that TBX5 mRNA expression level in heart tissues of individuals carrying CT-TT genotypes was only 49.3%that of the CC genotype in CHD group. Later, luciferase reporter assay suggested that the negative regulations of miR-9 and miR-30a on the two alleles were quite different. Furthermore, qRT-PCR and Western blot indicated that the polymorphism change from C to T could enhance the two miRNAs’inhibition of TBX5 gene expression. In detail, both of TBX5 mRNA and protein were downregulated by miR-9, whereas miR-30a mainly inhibited TBX5 translation.In order to identify more miRNAs targeting TBX5 3’UTR, we firstly chose miRNAs according to the prediction outcomes of bioinformatics, and then conducted co-transfection of miRNAs expression vector and Iuciferase based reporter vector in HEK 293T. It was found that miR-10a and miR-10b could significantly downregulate Iuciferase activity. The following qRT-PCR and Western blot confirmed that although miR-10a and miR-10b had no effect on TBX5 mRNA expression in HEK 293T, they could significantly downregulate TBX5 protein expression. Conversely, miR-10a/10b inhibitors could upegulate TBX5 expression at the translation level.With TBX5 binding sites located in the promoter region, ANF is considered as one of TBX5 downstream target genes. Therefore, we then determined the effect of miR-10a/10b on the expression of ANF. Luciferase activity of ANF promoter based pGL3-Basic vector was significantly decreased when co-transfected miR-10a/10b in HEK 293T, while it was not the case with Hela in which TBX5 expression was in low level. Consistent with the luciferase activity changes, the expression of ANF mRNA was downregulated in HEK 293T with overexpression of miR-10a/10b.In conclusion, we identified that TBX5 3’UTR rs6489956 C>T polymorphism could significantly increase the susceptibility of CHD in Chinese Han population for the first time, and that the negative modulation of miR-9 and miR-30a on T allele was more obvious, resulting in differential expression of TBX5. Meanwhile, we discovered that miR-10a and miR-10b could inhibit TBX5 expression at the translation level, and then downregulate ANFtranscription through directly targeting TBX5 3’UTR.