MiR4496 Negative Feedback On The Regulation Of Atrial Natriuretic Peptide By Glucocorticoid

NPPA gene encodes the atrial natriuretic peptide(ANP),which is a hormone synthesized and secreted by the atrium.ANP has natriuretic,diuretic,relaxes blood vessels,and inhibits the renin-angiotensin-aldosterone system(RAAS).ANP plays an important role in the regulation of the cardiovascular system.In our previous studies,we found that the NPPA gene polymorphism rs5063 SNP is associated with the occurrence of atrial fibrillation,we also reported that the second mutation of the NPPA gene,p.Ile138 Thr,and verified that the mutation of NPPA caused cardiac structural remodeling by inducing myocardial inflammation and fibrosis,and triggered atrial fibrillation.Based on this,this article will further study the expression regulation of NPPA gene.microRNA is a non-coding small moleculeRNA which discovered in recent years,mature miRNA mainly binds to the 3'UTR region of the target gene,inhibits the translation of the target gene or directly induces degradation of mRNA,and regulates cell proliferation.So far,three miRNAs,miR425,miR155,and miR105 have been reported to regulate NPPA at the transcriptional level,which inhibit the expression of NPPA by targeted binding to the NPPA 3'UTR region.In our previous work,we found a G>T change at position 133 of the NPPA gene in a patient with heart disease and atrial fibrillation.This site is located in the 3'UTR region of NPPA.The site may affect the transcription or translation of the NPPA gene,and there may be potential binding miRNAs which regulate the expression of NPPA.We used the miRNA database to predict the miRNAs at 20 bases before and after the 133 site in the 3'UTR region of NPPA,and learned that the site was in the seed sequence of the miR4496.We designed a double luciferase experiment on this segment of NPPA 3'UTR region and miR4496.The results showed that miR4496 significantly reduced the fluorescence intensity of the reporter gene in NPPA 3'UTR region.At the same time,we selected HEK293 T cells with high background expression of miR4496,and overexpressed miR4496,q RT-PCR and western blotting technology detectd the expression of NPPA mRNA and protein levels,respectively.To further verify the regulation of miR4496 on the NPPA gene,we overexpressed miR4496 in HEK293 T cells,Cell Counting Kit-8(CCK-8)measured the cell proliferation ability,and then added 8-Br-c GMP and IBMX respectively to perform ANP action supplementation and inspected the cell proliferation after stimulation.The results showed that in HEK293 T,NPPA is the target gene of miR4496,and miR4496 regulated the expression of NPPA gene not at the transcriptional level but at the post-transcriptional level.At the same time,miR4496 may promote the proliferation in HEK293 T cells by inhibiting the NPPA-dependent c GMP pathway,indicating that miR4496 inhibit the expression of NPPA.Glucocorticoid(GC)is a common drug that suppresses inflammation in the clinical tratement.It is widely used to prevent inflammation-related diseases such as inflammatory enteritis and rheumatoid arthritis.There are many mechanisms of glucocorticoid action,and it is considered to be a common mechanism by binding to glucococorticoid receptor(GR).As a common ligand-dependent transcription factor,GR regulates the transcription of target genes in the nucleus by binding to the promoter region of the target gene or interacting with other transcription factors.In addition,in recent years,it has also been found that glucocorticoids can also regulate the non-coding proteins,such as miRNAs.At the same time,studies shows that the glucocorticoid response element(GRE)is located in the promoter region of the NPPA gene and regulates the expression of NPPA.Therefore,we examined the expression regulation of miR4496 participation by GC.We used PROMO and LASAGNA-Search 2.0 software to predict the transcription factor of the miR4496 promoter region and found that GR may bind to the miR4496 promoter region.To verify the regulatory effect on miR4496,we treated HEK293 T cells with DEX and q RT-PCR detected the expression level of mature miR4496.Then,the cells were pretreated with glucocorticoid receptor inhibitor RU486 for 2 h,and then treated with dexamethasone(DEX).QRT-PCR verified the expression of miR4496 mRNA level.The results showed that glucocorticoid significantly up-regulated the expression of miR4496 at 4 h,GR participate in the regulation as a transcription factor.In order to verify the regulation of NPPA gene expression by glucocorticoids,we selected HEK293 T cells with higher background levels of NPPA and treated them with DEX to detect the expression of NPPA at the mRNA and protein levels.We used PROMO and LASAGNA-Search 2.0 technology software to predict the transcription factor of the NPPA promoter region.We used RU486,a glucocorticoid receptor inhibitor,to pretreat the cells.After DEX treatment,q RT-PCR was used to detect the NPPA mRNA level,western blotting detected the expression of ANP protein.We verified that glucocorticoids significantly increased the expression of NPPA at the mRNA and protein levels in a GR-dependent manner,and showed a time-dependent increase.In summary,we have discovered a brand new microRNA,microRNA4496,which can suppress the expression of NPPA gene at the post-transcription level,and verified that glucocorticoid significantly upregulates the expression of microRNA4496 at 4h,and the glucocorticoid receptor GR as a transcription factor participates in the regulatory process.At the same time,glucocorticoid up-regulates the expression of NPPA gene in a time-dependent manner,suggesting that GC,microRNA4496 and NPPA may constitute a regulatory loop with negative feedback.The study provide new targets for the steady state of the cardiovascular system regulated by ANP and for the treatment of atrial fibrillation and heart failure.