| Cardiovascular disease,one of the greatest threats to human health,has high morbidity and mortality.the development of cardiovascular disease(CVD)is very complex,the relevant mechanism of pathogenesis is no perfect theory in recent paper.The study found that myocardial hypertrophy and myocardial fibrosis were often accompanied by myocardial remodeling in the process of cardiovascular disease,which lead to cardiac function abate,heart blood supply insufficiency,heart failure and eventually death.While small noncoding RNAs(mi RNA,micro RNA)were involved in cardiovascular disease,especially in the process of myocardial fibrosis development.They combined to the 3 ’UTR of target genes and promoted the degradation of target genes m RNA,so as to exert mi RNA biological effect.Aim : The roles of mi R-199a-5p and mi R-199a-3p in cardiac fibrosis were not well illustrated.This paper aimed to establish the mouse model and cell model of Ang-Ⅱ induced cardiac fibrosis and investigated the protential target genes of mi R-199a-5p and mi R-199a-3p.Methods: 1.Animal model of Ang-Ⅱ capsule osmotic pump(1.46 mg/kg,2 weeks)to establish the animal model of myocardial fibrosis in C57BL/6 mice and to determind the expression of cardiac fibrosis related genes,mi R-199a-5p and mi R-199a-3p.2.The small animal ultrasound images and masson staining to detect the degree of myocardial fibrosis in mice and observe the changes of cardiac function.3.The original generation separation C57BL/6 mice myocardial fibroblasts.4.Dual luciferase reporter was performed to verify the interaction between mi R-199a-5p and the 3’UTR of Sirt1,mi R-199a-3p and the 3’UTR of Smad1.5.RT-q PCR and Western blot were performed to detect fibrosis related genes and protein expression in fibroblasts transfected with mi R-199a-5p and Sirt1 si RNA.6.RT-q PCR and Western blot were performed to detect fibrosis related genes and protein expression in fibroblasts transfected with mi R-199a-3p and Smad1 si RNA.7.Recombinate adenovirus of Sirt1 upregulated the expression of Sirt1 in cardiac fibroblasts and fibrosis related genes were detected.8.Ang-Ⅱ activate NF-κB and Smad3 signal pathways in cardiac fibroblasts,JSH23 and QNZ were used to block NF-κB signaling pathways,SIS3 and Naringenin were used to block Smad3 signaling pathway.9.RT-q PCR was performed to detect mi R-199a-5p,-3p and mi R-199 a transcript expression in fibroblasts transfected with Srf si RNA.Result: 1.mice in Ang-Ⅱ infusion group were found cardiac hypertrophy,appeared in the myocardial function abate,ventricular wall thickening and celluar interstitial collagen deposition.Cardiac fibrosis related genes,mi R-199a-5p and mi R-199a-3p were up-regulated.Both of them might participate in the myocardial fibrosis and cardiac remodeling.2.Ang-Ⅱ could effectively induce mice myocardial fibrosis in fibroblasts,and the expression of fibrosis related genes increased consistency.3.The overexpression of mi R-199a-5p and silence Sirt1 could increase consistency fibrosis related genes m RNA and protein level.At the same time using the Sirt1 adenovirus could improve Sirt1 expression in cells,and reduce pro-fibrosis effect of mi R-199a-5p.4.Sirt1 was confirmed the target gene of mi R-199a-5p and mi R-199a-5p regulated the expression of Sirt1 in transcript.5.Smad1 was confirmed the target gene of mi R-199a-3p and mi R-199a-3p regulated the expression of Smad1 in transcript.6.Ang-Ⅱ could activate the p65 NF-κB and Smad3 signaling pathway and upregulate the expression of mi R-199a-5p,-3p and mi R-199 a transcript.Using JSH23 and QNZ to block the NF-κB signaling pathway or using SIS3 and Naringenin to block Smad3 signaling pathway could reduce the mi R-199a-5p,-3p and mi R-199 a transcript expression.7.mi R-199a-5p could promote phosphorylated and acetylated Smad3 protein expression and activation of Smad3 signaling pathway by inhibiting Sirt1 expression,thus promoting cardiac fibrosis.8.mi R-199a-3p could promote Smad3 protein phosphorylated and activation of Smad3 signaling pathway by inhibiting Smad1 expression,thus promoting cardiac fibrosis.9.Ang-Ⅱ could increase the expression of Srf,to silence Srf could reduce mi R-199a-5p,-3p and mi R-199 a transcript expression.Conclusion: 1.Ang-Ⅱ infusion animal models of myocardial fibrosis in mice were successfully built,and the expression of mi R-199a-5p and mi R-199a-3p in myocardial fibrosis in mice express significant increased in heart tissues.2.The expression of mi R-199a-5p and mi R-199a-3p in Ang-Ⅱ mice model expressed were significant increase.3.Sirt1 was mi R-199a-5p potential target genes and Sirt1 mediated mi R-199a-5p promoted myocardial fibrosis in mice.Smad1 is mi R-199a-3p potential target genes and Smad1 mediated mi R-199a-3p promoted myocardial fibrosis in mice.4.mi R-199a-5p and mi R-199a-3p could inhibit the corresponding target genes Sirt1 and Smad1 expression and activated the Smad3 signaling pathways.5.Ang-Ⅱ activated mouse myocardial fibroblasts NF-κB and Smad3 signaling pathway,and mediated mi R-199a-5p,-3p and mi R-199 a transcript expression. |
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