Study Of The Role Of Transcription Factor NKX2-5 In The Development Of Heart

Objectives:NKX2-5 is a key transcription factor that regulates embryonic heart development,and it also plays an important role in maintaining normal contractile function and electrophysiological activities of the heart.The abnormal function of NKX2-5 leads to abnormal cardiac structure and function.At present,the specific molecular mechanism of NKX2-5 regulating the development of the heart and the differentiation and function of cardiomyocytes remains to be analyzed.This project aims to study the molecular mechanism of NKX2-5 regulating the development of the heart and the differentiation and function of the cardiomyocyte,as well as the correlation between gene mutation of NKX2-5 and the occurrence of the human congenital atrial septal defect.Methods:(1)DNA from peripheral blood of 30 patients with ASD and 30 healthy controls was extracted,and the full-length of NKX2-5 was sequenced by using Illumina sequencing technology.(2)The shRNA lentiviral vector was constructed to knock down the Nkx2-5 in the rat cardiomyocyte cell line H9c2.And the proliferation and migration of H9c2 and the expression of genes related to the differentiation and development of cardiomyocytes(Actnl,Tnnt2,Tbx5,Gata4,Hand2,Fgf10)were detected.(3)RNA-seq technology was used to analyze the changes in the gene expression profile of H9c2 cells after knocking down Nkx2-5.Bioinformatics was used to analyzes the signaling pathways enriched by the differentially expressed genes to find the key genes and signaling pathways regulated by the Nkx2-5.(4)qRT-PCR was used to verify the differentially expressed genes,and western blot technology was used to detect the activation status of the signaling pathway enriched by the differentially expressed genes.(5)TGF-? signaling pathway inhibitor and MAPK signaling pathway inhibitor inhibits the TGF-? signaling pathway and MAPK signaling pathway of H9c2 cells,respectively,and detect the proliferation ability of H9c2 cells and the expression of genes related to the differentiation and function of the cardiomyocyte.Results(1)Single nucleotide mutations were detected in NKX2-5 of patients with ASD.The mutation sites included rs2277923,rs3131915,173233524,173233520,173233516,and rs703752.The frequencies of mutations were 83.3%,13.3%,70%,26.7%,and 3.3%,and 10%,respectively.Single nucleotide mutations were detected in NKX2-5 of the healthy controls.The mutation sites include rs2277923,rs3131915,173233524,173233520,173233516,and rs703752.The frequencies of mutation were 80%,13.3%,80%,16.7%,10%and 13.3%,respectively.In the patients group and the healthy controls group,the OR values(95%CI)of the above single nucleotide site variations were 1.25(0.34-4.64),1,0.58(0.18-1.91),1.82(0.52-6.32),0.31(0.03-2.84)and 0.72(0.15-3.57),respectively.(2)The Knockdown of Nkx2-5 in H9c2 cells resulted in decreased cell proliferation,enhanced cell migration,and decreased expression of Tnnt2,Actn1,Gja1,Tbx5,Gata4,and Hand2.(3)The results of RNA-seq suggested that the knockdown of Nkx2-5 leads to changes in the expression of downstream genes.GO enrichment analysis suggested that the differentially expressed genes are enriched in extracellular space,extracellular matrix,calcium-dependent phospholipid binding,and calcium ion-dependent regulation of exocytosis,cardiac endothelialization,cardiac HIS bundle development,and cardiomyocyte proliferation and other biological processes.pathway enrichment analysis suggested that the differentially expressed genes are enriched in TGF-? signaling pathway,hypertrophic cardiomyopathy,dilated myocardium Disease,arrhythmia right ventricular cardiomyopathy-related pathway.(4)The results of RNA-seq suggested that the knockdown of Nkx2-5 changes the expression of downstream miRNAs.After target genes were predicted,GO enrichment analysis was performed on the target genes of these differentially expressed miRNAs.The results suggested that the target genes of the differentially expressed miRNAs are enriched in a variety of biological processes,including gene transcriptional regulation,redox reactions,cell signal transduction,cell differentiation,apoptosis,cell proliferation,protein phosphorylation,proteolysis,intracellular signal transduction,protein ubiquitin gene expression.And the molecular functions played by those target genes include protein binding,metal ion binding,ATP binding,homologous protein binding,homologous domain protein dimer activity,DNA binding,RNA binding,zinc ion binding,calcium ion binding.Pathway enrichment analysis suggested that the target genes of the differentially expressed miRNAs are enriched in MAPK signaling pathway,FoxO signaling pathway,mTOR signaling pathway,p53 signaling pathway,Ras s signaling pathway and Sphinolipid signaling pathway.(5)The results of qRT-PCR suggested that the knockdown of Nkx2-5 changes the expression of genes related to cell proliferation,migration and the differentiation and function of the cardiomyocyte,namely TGF-?2,Id2,Wt1,Cacnalg,Hey1,Tnnt2,Actn1,Gja1,Tbx5,Gata4,Myl2,Hand2,Fgf10 and miRNA1.The results of Western blot suggested that the knockdown of Nkx2-5 leads to inhibition of the MAPK signaling pathway.(6)Inhibition of the TGF-? signaling pathway leads to decreased proliferation of H9c2 cells.Inhibition of the TGF-?signaling pathway leads to decreased expression of Tnnt2,and increased expression of Actnl,Gjal,Gata4,Hand2,Id2,Wnt4 and Xdh.(7)Inhibition of the MAPK signaling pathway leads to decreased proliferation of H9c2 cells.Inhibition of the MAPK signaling pathway leads to increased expression of Tnnt2,Wnt4,and Lrp2,and decreased expression of Actnl,Gjal,Tbx5,Hand2,Wtl,Heyl,Xdh,Cacna1g,Syt1,Pou5f1,and Xdh.Conclusions:1.There are single nucleotide site variations in NKX2-5 of patients with ASD.Those site variations may be related to the occurrence of ASD.2.Nkx2-5 promotes the proliferation of rat cardiomyocyte H9c2,inhibits cell migration,and regulates the expression of genes related to the differentiation and function of the cardiomyocytes3.The activation of the TGF-? signaling pathway promotes the proliferation of rat cardiomyocyte H9c2 and regulates the expression of genes related to the differentiation and function of the cardiomyocytes.4.The activation of the MAPK signaling pathway promotes the proliferation of rat cardiomyocyte H9c2 and regulates the expression of genes related to the differentiation and function of the cardiomyocytes.5.The Transcription factor NKX2-5 may regulate the proliferation,migration,and the differentiation and function of the cardiomyocytes through the TGF-?2,Id2,Wt1,Cacna1g,Hey1,Tnnt2,Actn1,Gja1,Tbx5,Gata4,Myl2,Hand2,Fgf10,miRNA1,TGF-? signaling pathway,and MAPK signaling pathway.