MiR-21 Promotes Myocardial Fibrosis By Targeting Jagged1

Chapter 1 IntroductionMyocardial infarction is the main cause of sudden death.Cardiac remodeling characterized by cardiac hypertrophy and interstitial fibrosis occur due to abnormal activation of myocardial fibroblasts in the late repair of myocardial infarction.Studies have shown that the five-year survival rate of patients who suffered from heart failure due to ventricular remodeling is less than 35%.Therefore,how to prevent the ventricular remodeling and interstitial fibrosis during the repair period of myocardial infarction in time,which is the main difficulty in the treatment of acute myocardial infarction.Myocardial fibrosis is an important pathological change after myocardial infarction.Excessive myocardial fibrosis will lead to ventricular dilatation,myocardial expansion in the infarction area,heart failure and eventually death.Therefore,the search of new stable and reliable biomarkers for myocardial fibrosis,early diagnosis of myocardial fibrosis,and further study of its regulatory mechanism in myocardial fibrosis is expected to improve the overall treatment level of ischemic heart disease.MicroRNA is a negative regulator non-coding RNA that regulates gene expression by enhancing mRNA degradation or inhibiting mRNA translation.Among several micrornas involved in myocardial fibrosis,miR-21 plays a significant role in fibrosis in multiple organs such as liver,kidney and lung.A recent study reported that miR-21 promotes myocardial fibrosis after myocardial infarction through Smad7 ligand,which found that cell transformation growth factor-1(TGF-?1)signaling is the main regulatory pathway for myocardial repair and fibrosis after myocardial infarction.After myocardial infarction,TGF-?1 was significantly up-regulated in myocardial fibroblasts,and the transformation of myocardial fibroblasts to myofibroblasts was promoted through the TGF-?1/Smad3 signaling pathway.Given that miRNA as a target regulates various physiological and pathological processes,we hypothesized that miR-21 may be involved in myocardial fibrosis by targeting other signaling pathways.Interestingly,in breast cancer cells,the Notch ligand Jagged 1 is a direct target of miR-21.However,the mechanism of miR-21 and Notch signaling pathways in regulating myocardial fibrosis is not clear.Therefore,this study aimed to explore whether miR-21 regulates myocardial fibrosis through the Notch/Jagged1 signaling pathway.Chapter 2 miR-21 promotes myocardial fibrosis induced by TGF-?1Objective: To explore how miR-21 mediating TGF-?1 to induce myocardial fibrosis.Adenovirus vectors for inhibiting the expression of miR-21 were constructed to investigate whether miR-21 regulates myocardial fibrosis by promoting the TGF-?1 signaling pathway.Methods: Rat cardiac fibroblasts were treated with TGF-?1 and the effects of miR-21 inhibition on TGF-?1 were observed.Western blotting and qPCR were used to analyze the effects of inhibition of miR-21 binding TGF-?1 on the protein level and RNA level of each marker protein.CCK-8 and Transwell assay were used to determine the effects of inhibiting miR-21 on the proliferation and invasion of cardiac fibroblasts mediated by TGF-?1.Results: The expression of miR-21 was significantly increased after the treatment of CFB with TGF-?1,indicating that the expression of miR-21 was induced by myocardial fibrosis.The CCK8 experiment showed that the miR-21 activity of cardiac fibroblasts was decreased,and the proliferation capacity of cardiac fibroblasts was impaired.At the same time,decreased miR-21 activity can inhibit the proliferation performance of cardiac fibroblasts induced by TGF-?1.The Transwell test showed that the activity of miR-21 in cardiac fibroblasts was decreased and the invasiveness of cardiac fibroblasts was decreased.At the same time,the activity of miR-21 was decreased and the invasiveness of cardiac fibroblasts induced by TGF-?1 was inhibited.Western blotting and qPCR analyzed the inhibition of TGF-?1 induced CMT by lowering miR-21 activity at the protein level and RNA level respectively.Conclusion: miR-21 promotes myocardial fibrosis induced by TGF-?1.Inhibition of miR-21 expression could significantly improve myocardial fibrosis.Moreover,miR-21 accelerates fibrosis by promoting the expression of TGF-?1.Chapter 3 miR-21 targets on Jagged1 and promotes myocardial fibrosis Objective: To verify whether miR-21 can bind to Jagged1 firstly.Furthermore,it was verified whether miR-21 pathway acting on myocardial fibrosis was induced by Jagged1 ligand mediated TGF-?1.Methods: Luciferase experiment was used to verify whether miR-21 could bind to Jagged1.Western blotting analysis was used to study the effect of TGF-?1 and miR-21 inhibitors on the level of myofibroblast marker protein in rat CFs.The effect of TGF-?1 and miR-21 inhibitors on m RNA levels of myofibroblast markers in rat CFs was studied by q PCR.The relationship between miR-21,TGF-?1 and Jagged1 was determined by CCK8 and Transwell experiments.Results: The dual-luciferase reporter gene experiment verified that miR-21 target binding to Jagged1 3'-UTR.After treatment with adenovirus inhibiting miR-21,Jagged1 expression was significantly increased after inhibition of miR-21 activity.Western blotting and q PCR confirmed the inhibition of Jagged1 by TGF-?1 by decreasing the miR-21 activity of cardiac fibroblasts from protein and RNA levels,respectively.The CCK8 experiment showed that the inhibition of miR-21 inhibitor on the proliferation of cardiac fibroblasts was completely eliminated by reducing the expression of Jagged1.The Transwell experiment showed that the inhibition of miR-21 inhibitor on the invasiveness of cardiac fibroblasts was completely eliminated by reducing the expression of Jagged1.Western blotting and q PCR demonstrated from the protein level and RNA level respectively that the reduced expression of Jagged1 could completely inhibit the inhibition effect of miR-21 inhibitors on CMT.Conclusion: miR-21 could target and bind to Jagged1 3'-UTR.By lowering the activity of miR-21,TGF-?1 inhibits Jagged1 and promotes myocardial fibrosis.Chapter 4 miR-21 was verified to promote myocardial fibrosis in vivo Objective: To explore how miR-21 can target Jagged1 pathway and promote myocardial fibrosis in rat heart failure model.Methods: Male SD rats were used to establish a rat model of heart failure by ligation of 8-0 nylon line at 3-5mm LAD.The degree of myocardial fibrosis was detected by Masson staining.The effect of miR-21 targeting on Jagged1 of the rats left ventricular function was detected by cardiac ultrasound.Results: Cardiac dysfunction was improved after inhibition of miR-21 and deteriorated after reduction of Jagged1.At the same time,after the rats were treated with both miR-21 inhibition and Jagged1 reduction,the two effects offset each other.Compared with the control group,both EF and FS increased in the miR-21 inhibition group,and LVEDV and LVESV decreased,significantly improving the recovery of cardiac function.However,the inhibition effect of miR-21 was eliminated after reduction of Jagged1,and left cardiac function deteriorated significantly.Masson staining showed that cardiac insufficiency was improved after being treated with miR-21 inhibition and reduced postoperative deterioration of Jagged1.At the same time,the effects of both miR-21 inhibition and Jagged1 reduction on the rats cancel each other out.Conclusion: miR-21 could promote myocardial fibrosis by targeting Jagged1 ligand in vivo.