Screening And Functional Study Of Differentially Expressed Genes Related To Heart Development Downstream Of TBX1 Gene

Background and Objectives: Congenital heart disease(CHD)is the most common birth defect in newborns and the main cause of perinatal and child mortality.Studies have confirmed that CHD is mainly caused by the combination of genetic and environmental factors during the embryonic period,of which genetic factors play an important role.However,the mechanism of the relationship between cardiac development-related genes and signaling pathways with CHD is still unclear.The development of the heart is a more complex biological process regulated by multiple genes.Studies have shown that transcription factors can directly or indirectly regulate the expression of target genes,affect the migration,proliferation and differentiation of cardiac muscle cells,and thus affect the occurrence and development of congenital heart disease.TBX1 gene is a transcription factor of the T-box gene family.It is expressed early in embryonic development and is closely related to heart development.It is one of the pathogenic genes of CHD.At present,the research on TBX1 gene is limited to nucleotide mutations or gene deletions and duplications in the coding region of TBX1 gene.However,there are few studies on the abnormal regulation of TBX1 gene expression.In our previous study,we found that the T350 M polymorphism of Tbx1 gene has a significant correlation with Tetralogy of Fallot(TOF),which may be the genetic susceptibility gene of TOF.Our research team found for the first time that the expression level of ACTC1 gene in the myocardium of CHD patients decreased.This abnormality can induce excessive apoptosis of cardiomyocytes during embryonic development,thus leading to the occurrence of human CHD.Our team further found that HOXA3 transcription factor binding site existed in the upstream regulatory sequence of ACTC1,and the interaction between HOXA3 and TBX1 was involved in heart development,which was closely related to the occurrence of CHD.During early embryonic development,the loss of apoptosis-related cardiomyocytes is one of the key factors leading to CHD.The gene regulation mechanism targeting cardiomyocyte apoptosis may be the key to influence the occurrence and development of CHD.Its exact mechanism urgently needs further study.TBX3 gene participates in the development of multiple organs in the early embryo.Moreover,studies have shown that TBX3 gene can inhibit the expression of cell cycle regulatory protein P21,and thus play a role in regulating cell cycle,inhibiting apoptosis and promoting proliferation.As an inhibitor of the CDK family,P21 gene is essential for regulating cell cycle and cell proliferation,but the role of P21 gene in cardiomyocytes has not been reported.Therefore,it is necessary to explore the differentially expressed genes related to cardiac development of TBX1 gene’s downstream,and whether TBX3 gene acts as a downstream signaling molecule of TBX1 gene to regulate the biological function of cardiomyocytes through P21 gene.Further elucidating the molecular mechanism of TBX1 gene involved in cardiac developmental signaling pathways will help to explore new prenatal diagnostic targets for congenital heart disease and provide new theoretical basis for targeted treatment of congenital heart disease.Methods: Part 1: Transfect rat cardiomyocyte H9C2 cells with TBX1 siRNA and use high-throughput gene chip technology to screen differentially expressed genes related to heart development of TBX1 gene downstream,Perform GO enrichment analysis and KEGG database Pathway enrichment analysis.Real-time PCR and ELISA were used to detect the expression of TBX3 gene in CHD myocardial tissues.Part 2: Establish TBX3 gene silenced and over-expressed H9C2 cells,and detect TBX3 gene expression by Real-time PCR and Western blot.Secondly,CCK-8 and Ki67 immunofluorescence staining were used to detect cell activity;flow cytometry was used to detect cell cycle distribution,and Western blot was used to detect CyclinA,CyclinB1,CyclinD1 expression.Then use Hoechst33342 staining and Annexin V/PI double staining to detect apoptosis,Western blot to detect apoptosis-related proteins cleaved caspase-9,cleaved caspase-3,cleaved PARP,Bax,Bcl-2 Expression.Jc-1 method was used to detect mitochondrial membrane potential,kit method was used to detect mitochondrial ATPase activity and ROS content.then real-time PCR and Western blot were used to detect P21 gene expression.Ch IP experiment verified that TBX3 gene interacted with P21 DNA.Finally,by simultaneously silencing TBX3 gene and P21 gene,the effects of P21 gene on TBX3 gene silencing-induced P21 gene expression and cell proliferation were examined.Results:Part 1: 1.There were 995 differentially expressed genes of TBX1 gene downstream,of which 240 genes were up-regulated and 755 genes were down-regulated.2.GO enrichment analysis showed that the biological processes that up-regulate differentially expressed genes with a high degree of enrichment are mainly glycolytic process,cholesterol biosynthetic process,carbohydrate phosphorylation,cell migration,and lipid phosphorylation.Biological processes related to the heart include heart valve morphogenesis,heart morphogenesis.Related differential genes include Tgfb2、Sox9、Tab1;The biological processes that down-regulate differentially expressed genes with high enrichment are mainly: cell division,chromosome segregation,mitotic cell cycle and response to virus.Biological processes related to the heart include heart morphogenesis,embryonic heart tube morphogenesis,embryonic heart tube left/right pattern formation,heart rate regulation,and heart development.Related differential genes include TBX3、Cited2、Tead2、Pln、Casq2、Dmd、Ift122、Kcne2、Dchs1、Nrp2、Gli2.3.KEGG database Pathway enrichment analysis shows that the statistically significant signal pathways in the differential genes are cell cycle,cytoplasmic DNA sensing pathway,meiosis,etc.4.TBX3 mRNA and protein expression levels were significantly reduced in CHD cardiac tissue samples.Part 2: 1.Silenced TBX3 gene significantly inhibited H9C2 cell proliferation and Ki67 protein expression.Over-expressed TBX3 gene had the opposite effect.2.Silenced TBX3 gene significantly increased the number of H9C2 cells in G1 phase,reduced the number of H9C2 cells in S phase,and inhibited the expression of Cyclin D1,Cyclin A,and Cyclin B1.Overexpression of TBX3 gene could enhance the expression of cyclin and had no significant effect on cell cycle distribution.3.TBX3 gene silenced H9C2 cells showed typical apoptotic morphological characteristics,including nuclear fragmentation and chromatin concentration,and the percentage of apoptotic cells increased significantly.cleaved caspase-9,cleaved caspase-3,cleaved PARP,Bax expression up-regulation,Bcl-2 expression was down-regulated.Overexpression of TBX3 gene had no significant effect on the apoptotic state of the cells.4.Silenced TBX3 gene can significantly down-regulate mitochondrial membrane potential and ATPase activity of H9C2 cells,increase ROS content,increase cytochrome C content in cytoplasm,and reduce cytochrome C content in mitochondria.5.TBX3 gene silencing significantly increased the expression levels of P21 mRNA and protein in H9C2 cells,and there was targeted binding between TBX3 gene and P21 DNA.6.Simultaneous silencing of TBX3 gene and P21 gene can significantly reduce the expression of P21 gene in H9C2 cells,and can obviously restore the decline of H9C2 cell proliferation ability induced by TBX3 gene silencing.Conclusion:1.Upregulated differentially expressed genes of TBX1 gene downstream are related to biological processes involved in biosynthesis and phosphorylation,down-regulated genes are related to cell division,cell cycle,and response to certain biochemical substances.Biological processes related to the heart include heart morphogenesis,embryonic heart tube morphogenesis,embryonic heart tube left/right pattern formation,heart rate regulation,and heart development,etc.2.TBX3 gene is a differentially expressed gene related to heart development of TBX1 gene downstream.3.Low expression of TBX3 gene is associated with CHD.4.TBX3 gene silencing inhibits the proliferation of H9C2 cardiomyocytes,blocks the cell cycle in the G1 phase.5.TBX3 gene silencing induces H9C2 cardiomyocytes mitochondrial dysfunction,and promotes apoptosis.6.P21 gene is a direct target gene of TBX3 gene downstream.7.Silencing TBX3 gene inhibits the proliferation of H9C2 cardiomyocytes by targeting the regulation of P21 gene expression.