| Congenital heart disease(CHD)is a type of cardiovascular disease caused by structural abnormalities of the heart and blood vessels during embryonic development.It is also the most common type of birth defect in China.The occurrence and development of congenital heart disease are the result of the joint action of heredity and environment.GDF1 plays an important role in the establishment of bilateral axial asymmetry of embryos and heart circularization.GDF1 mutation is closely related to the occurrence of CHD,but the research on the transcriptional regulation mechanism of GDF1 is rarely reported.GDF1 is encoded by a conserved double cistron mRNA,which contains two coding regions,GDF1 and Cersl,of which Cersl is located in the upstream of GDF1.Our laboratory has previously confirmed that the upstream and downstream region of ATG(+1)of Cers1 translation initiation site contains the basic promoter sequence of GDF1.We used the transcription factor binding software PROMO and JASPAR to perform bioinformatics analysis on the basic sequence of GDF1 promoter and found three potential NKE sites,which are binding elements of NK homologous domain proteins on DNA.Nkx2.5,as one of the members of NK homologous and heteromorphic domain protein nk-2 necessary for the development of myogenic pedigree,plays an important role in the development of heart,and its mutation can lead to a variety of CHD phenotypes.Therefore,we focused on the transcription factor Nkx2.5,and further identified the regulation of Nkx2.5 on GDF1 through experiments.We used the pcDNA3.1-myc-His empty vector and the pcDNA3.1-myc-Nkx2.5 expression plasmid to co-transfection HEK293ET cells with the pGL3-GDF1 promoter reporter plasmid,and detected the activity of the GDF1 promoter in the dual-fluorescent reporter gene experiment,and found that the overexpression of Nkx2.5 activated the activity of the GDF1 promoter.After transfection of HEK293ET cells with pcDNA3.1-myc-His empty vector and pcDNA3.1-myc-Nkx2.5 expression plasmid for 24h,real-time PCR results showed that overexpression of Nkx2.5 significantly increased the level of GDF1 mRNA,and Western Blot results showed that overexpression of Nkx2.5 significantly increased the level of GDF1 protein.After infection with myocardial cell H9c2 for 24h,with recombinant adenovirus control and recombinant adenovirus Ad-Nkx2.5,western Blot results showed that overexpression of Nkx2.5 significantly increased GDF1 protein level.The zebrafish Dvrl gene is a homologous gene of GDF1 in mammals.Subsequently,using the vertebrate model zebrafish as the model,we used morpholino technology to knock down the Nkx2.5 gene.The results of in situ hybridization and real-time PCR showed that Nkx2.5 knockdown significantly reduced the transcription level of Dvrl in zebrafish embryos,while the overexpression of Nkx2.5 reversed the down-regulation of Dvrl transcription level in zebrafish.These results suggest that Nkx2.5 can activate the expression of GDF1 at the transcription level.We further identified the binding site of Nkx2.5 in the promoter region of GDF1.Bioinformatics analysis revealed that three potential NKE loci were located at-517,-440 and-203 in the promoter region of GDF1.We constructed three potential NKE reporter plasmids using wild-type GDF1 basic promoter region-608/+71 as the skeleton,and then transfected HEK293ET and H9c2 cells with the constructed reporter plasmids.The detection of dual-fluorescence reporter gene activity showed that the point mutation at the binding site of-517 inhibited the activity of GDF1 promoter in the two kinds of cells significantly more than that in the control group,while the inhibition effect of the-440 or-203 site mutation was weak or not obvious.Therefore,we studied the effect of-517 locus on the regulation of GDF1 function by Nkx2.5.Nucleoprotein was extracted from HEK293ET cells transfected with pcDNA3,1-myc-Nkx2.5 as sample,biotin-labeled-517 wild-type and mutant double-stranded oligonucleotides were used as probes,and DNA pulldown analysis was performed.The results showed that the binding ability of the mutant-517 binding site to Nkx2.5 protein was significantly decreased compared with the wild-type-517 binding ability.Subsequently,The reporter plasmids of the wild-type GDF1 promoter and the mutation of-517 binding site,and the expression plasmid of pcDNA3.1-myc-nkx2.5 with different concentrations was used to co-transfect HEK293ET and H9c2 cells.The results of dual-fluorescence reporter gene detection showed that overexpression of Nkx2.5 in both cells led to a significant increase in dose-dependent activity of the wild-type GDF1 promoter,while the transcription activity of Nkx2.5 was reduced by about 70%without a dose-dependent increase after the-517 binding site mutation.HEK293ET cells were transfected with pcDNA3.1-myc-nkx2.5 and the chromatin immunoprecipitation was used to analyze the binding of NKX2.5 to GDF1 promoter in vivo.Real-time PCR results showed that-550/-276 region containing-517 site was significantly amplified.In summary,we found that the overexpression of Nkx2.5 activates the activity of GDF1 promoter and up-regulates the expression of GDF1.Nkx2.5 knockdown significantly reduced the transcription level of the mammalian GDF1 homologous gene Dvrl in zebrafish,while the overexpression of Nkx2.5 reversed this effect.Further mechanism studies found that Nkx2.5 can regulate GDF1 expression through-517 locus.These results indicate that GDF1 is a novel target gene of Nkx2.5.In addition,miRNA microarray analysis was conducted in our laboratory in the primary cultured rat granulosa cells with significantly increased progesterone synthesis before and after FSH stimulation.31 differentially expressed mimas were found,among which miR-132 was one of the genes with the most significant difference in the amplitude of FSH regulation.Therefore,we preliminarily explored the role of miR-132 in the synthesis of progesterone in granulosa cells mediated by follicle stimulating hormone(FSH).We treated primary granulosa cells with 50ng/ml FSH and conducted real-time PCR detection,and found that the expression level of miR-132 was significantly up-regulated.Primary granulosa cells were infected with recombinant adenovirus control and recombinant adenovirus Ad-miR132.Real-time PCR results showed that the expression level of miR-132 was significantly increased after the infection of Ad-miR132.The primary granulosa cells were infected with the recombinant adenovirus control and Ad-miR132 and then treated with FSH.The radioimmunoassay results showed that FSH-mediated progesterone synthesis in granulosa cells was significantly increased after the infection with Ad-miR132.It has been reported that Foxo3a plays an important role in follicular development,and Foxo3a is known to be a downstream target gene of miR-132.The expression of Foxo3a in granulosa cells treated with FSH was detected by Western Blot,and the results showed that the expression of Foxo3a showed a gradually decreasing trend at different time points of FSH treatment,suggesting that miR-132 may negatively regulate the expression of Foxo3a in granulosa cells.The above results indicated that miR-132 promoted the synthesis of progesterone in granular cells mediated by FSH. |
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