Shujing Yuan1, Jingnan Liang2, Ming Zhang3, Jiening Zhu3, Rong Pan4, Hui Li3, Ni Zeng1, Yihong Wen1, Zhiyao Yi4, Zhixin Shan1,3. 1. School of Medicine, South China University of Technology, Guangzhou 510006, China. 2. Department of Pharmacy, Jiangmen Central Hospital, Jiangmen 529030, China. 3. Guangdong Cardiovascular Institute//Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China. 4. School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China.
Abstract
OBJECTIVE: To investigate the effect of circRNA_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_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_005647 in mouse CFs. Over- expression of circRNA_005647 was achieved in the CFs by infecting the cells with a recombinant circRNA_005647 adenovirus (rAd-circRNA_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_005647 and miR-27b-3p. RESULTS: CircRNA_005647 was up- regulated in the myocardium of Ang-Ⅱ- infused mice and in Ang-Ⅱ-treated mouse CFs (P < 0.01). CircRNA_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_ 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_005647. MiR-27b-3p obviously enhanced the fibrotic phenotype but reversed the inhibitory effect of circRNA_005647 on the expression of fibrosis associated genes in the CFs (P < 0.05). CONCLUSIONS: CircRNA_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 effect of circRNA_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_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_005647 in mouse CFs. Over- expression of circRNA_005647 was achieved in the CFs by infecting the cells with a recombinant circRNA_005647 adenovirus (rAd-circRNA_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_005647 and miR-27b-3p. RESULTS: CircRNA_005647 was up- regulated in the myocardium of Ang-Ⅱ- infused mice and in Ang-Ⅱ-treated mouse CFs (P < 0.01). CircRNA_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_ 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_005647. MiR-27b-3p obviously enhanced the fibrotic phenotype but reversed the inhibitory effect of circRNA_005647 on the expression of fibrosis associated genes in the CFs (P < 0.05). CONCLUSIONS: CircRNA_005647 is upregulated in cardiac fibrosis and inhibits the expression of fibrosis-related genes through sponging miR-27b-3p in mouse CFs.
Authors: Hernan A Bazan; Samuel A Hatfield; Aaron Brug; Ashton J Brooks; Daniel J Lightell; T Cooper Woods Journal: Circ Cardiovasc Genet Date: 2017-08