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Objective: To investigate the effect of circRNA&#95;005647 on fibrotic phenotype of mouse cardiac fibroblasts (CFs) and explore its mechanism., Methods: We used an angiotensin Ⅱ (Ang-Ⅱ) capsule pump (daily dose of 1.46 mg/kg for 2 weeks) to establish a mouse model of myocardial fibrosis in C57BL/6 mice. Masson staining was used to detect myocardial fibrosis in the myocardium. The expression of circRNA&#95;005647 in the myocardium of Ang-Ⅱ-infused mice and in Ang-Ⅱ-treated CFs were detected with real-time PCR. Actinomycin D and RNase R exonuclease digestion were used to test the stability of circRNA&#95;005647 in mouse CFs. Over- expression of circRNA&#95;005647 was achieved in the CFs by infecting the cells with a recombinant circRNA&#95;005647 adenovirus (rAd-circRNA&#95;005647), and the expressions of Col1a1, Col3a1 and Acta2 were detected in the cells with real-time PCR and Western blotting. Dual luciferase reporter assay, RNA antisense purification and RNA Pull down assay were performed to identify the interaction between circRNA&#95;005647 and miR-27b-3p., Results: CircRNA&#95;005647 was up- regulated in the myocardium of Ang-Ⅱ- infused mice and in Ang-Ⅱ-treated mouse CFs ( P < 0.01). CircRNA&#95;005647 was more stable than its host gene Myosin IXA ( Myo9a ) in response to actinomycin D ( P < 0.01) and RNase R exonuclease treatment. The expressions of fibrosis-associated genes was down-regulated in the CFs over-expressing circRNA&#95; 005647 ( P < 0.05). Dual luciferase reporter assay, RNA antisense purification and RNA Pull down assay revealed the interaction between miR-27b-3p and circRNA&#95;005647. MiR-27b-3p obviously enhanced the fibrotic phenotype but reversed the inhibitory effect of circRNA&#95;005647 on the expression of fibrosis associated genes in the CFs ( P < 0.05)., Conclusions: CircRNA&#95;005647 is upregulated in cardiac fibrosis and inhibits the expression of fibrosis-related genes through sponging miR-27b-3p in mouse CFs.

Objective: To investigate the role of miR-199a-3p in cardiac fibrosis and the potential target of miR-199a-3p., Methods: Cardiac fibroblasts were isolated from C57BL/6 mice and cultured. The miR-199a-3p mimic and Smad1 siRNA were transiently transfected into the cardiac fibroblasts via liposome. Dual luciferase reporter assay was performed to confirm the interaction between miR-199a-3p and the 3'-UTR of Smad1. The expressions of Smad1 and fibrosis-related genes at the mRNA and protein levels in the cells after miR-199a-3p mimic transfection were determined using RT-qPCR and Western blotting, respectively. The expressions of Smad1, Smad3 and fibrosis-related genes at the protein level in cells transfected with miR-199a-3p mimic and Smad1 siRNA were detected using Western blotting., Results: Over-expression of miR-199a-3p significantly increased the expression of cardiac fibrosis-related genes in cultured mouse cardiac fibroblasts. Dual luciferase reporter assay revealed the interaction of miR-199a-3p with the 3'-UTR of Smad1. The results of RT-qPCR and Western blotting confirmed that miR-199a-3p inhibited Smad1 expression at the post- transcriptional level. Transfection with miR-199a-3p mimic and siRNA-mediated Smad1 silencing consistently activated the Smad3 signaling pathway and enhanced the expressions of cardiac fibrosis-related genes in the cardiac fibroblasts., Conclusions: As the target gene of miR-199a-3p, Smad1 mediates the pro-fibrotic effect of miR-199a-3p by activating the Smad3 signaling in cultured mouse cardiac fibroblasts.

Hypertension is a main risk factor for atrial fibrillation, but the direct effects of hydrostatic pressure on the atrial fibrosis are still unknown. The present study investigated whether hydrostatic pressure is responsible for atrial fibrosis, and addressed a potential role of the Smad pathway in this pathology. Biochemical assays were used to study regulation and expression of fibrotic factors in spontaneously hypertensive rats (SHRs) and Wistar rats, and in cardiac fibroblasts (CFs) cultured under standard (0 mmHg) and elevated (20, 40 mmHg) hydrostatic pressure. Levels of atrial fibrosis and protein expression of fibrotic factors Col-1A1/-3A1, TGF-β1, and MMP-2 in SHRs' left atrial tissues were higher than those in Wistar rats. Exposure to elevated pressure was associated with the proliferation of CFs. The protein expression of Col-1A1/-3A1, TGF-β1, and MMP-2 in CFs was also up-regulated in a pressure-dependent manner. The proliferation of CFs and increased expressions of fibrotic markers induced by elevated hydrostatic pressure could be reversed by the Smad3 inhibitor naringenin. The activation of Smad3 pathway was also stimulated by elevated hydrostatic pressure. These results demonstrate that CF secretory function and proliferation can be up-regulated by exposure to elevated pressure, and that Smad3 may modulate CF activation induced by high hydrostatic pressure., (© 2017 Wiley Periodicals, Inc.)