circRNA-Zfp609对C2C12成肌细胞增殖和分化的影响

doi:10.16431/j.cnki.1671-7236.2022.12.003

Abstract

Abstract: 【Objective】 The purpose of the experiment was to explore the potential molecular mechanism of circRNA-Zfp609 regulating the proliferation and differentiation of C2C12 myoblasts.【Method】 The expression of circRNA-Zfp609 in mouse skeletal muscle tissues and C2C12 myoblasts were analyzed by RT-PCR and sequencing.The relative expression of circRNA-Zfp609 in heart, liver, spleen, lung, kidney, stomach, small intestine and skeletal muscle tissues of mice, in C2C12 myoblasts after 12, 24, 36 and 48 h proliferation and 0, 1, 3 and 5 d differentiation was detected by Real-time quantitative PCR.The relative expression of myogenin (MyoG) and myosin heavy chain (MyHC) in C2C12 myoblasts after 0, 1, 3 and 5 d differentiation was detected by Real-time quantitative PCR.The cells were interfered with the interfering expression vector (siRNA) of circRNA-Zfp609 and the effect of siRNA on the proliferation rate of C2C12 myoblasts was determined by CCK-8, and the effect of siRNA interference on the relative expression of circRNA-Zfp609, MyoG and MyHC were detected by Real-time quantitative PCR.The sites of muscle differentiation-related miRNAs on circRNA-Zfp609 were predicted by TargetScan 7.0 and miRDB softwares.The overexpression vectors of the screened miRNA were transfected into HEK293T cells, and the interaction between circRNA-Zfp609 and corresponding miRNA was verified by dual-luciferase reporter assay.According to the interaction of miRNAs with circRNA-Zfp609, the wild-type and mutant vectors of circRNA-Zfp609 were constructed and transfected into HEK293T cells, and the targeted relationship of circRNA-Zfp609 and miRNA was verified by dual-luciferase reporter assay.【Result】 The PCR and sequencing results showed that circRNA-Zfp609 was expressed in mouse skeletal muscle.circRNA-Zfp609 had the highest expression level in mouse skeletal muscle, and the expression in other tissues from high to low were kidney, lung, heart, liver, stomach, spleen and small intestine.Compared with 12 h, the relative expression of circRNA-Zfp609 was significantly increased at 36 and 48 h of C2C12 myoblast proliferation (P<0.05).Compared with differentiation day 0, the relative expression of circRNA-Zfp609 was significantly increased on days 1, 3 and 5 (P<0.05), and the relative expression of MyoG and MyHC were extremely significantly increased (P<0.01).Compared with the NC group, the proliferation rate and the relative expression of circRNA-Zfp609 of C2C12 myoblasts in the siRNA group were both extremely significantly decreased (P<0.01), and the relative expression of MyoG and MyHC were significantly decreased (P<0.05).There were 4 miRNAs (miR-150-5p, miR-327, miR-344g-3p and miR-615-5p) on circRNA-Zfp609 were related to muscle differentiation.circRNA-Zfp609 had the strongest adsorption capacity with miR-615-5p, and had a targeted binding effect.circRNA-Zfp609 could act as a molecular sponge to interact with miR-615-5p that regulated muscle differentiation.【Conclusion】 circRNA-Zfp609 was widely expressed in mouse tissues, with the highest expression level in skeletal muscle.circRNA-Zfp609 was differentially expressed in C2C12 myoblasts at different stages of proliferation and differentiation, circRNA-Zfp609 had 4 miRNAs related to muscle differentiation, and miR-615-5p had a targeting relationship with circRNA-Zfp609.The results could provide references for the research related to the growth and development of domestic animal skeletal muscle.

Key words: circRNA-Zfp609; C2C12 myoblasts; cell proliferation; cell differentiation; miR-615-5p

CLC Number:

S813.1

LIU Zhuang, HE Ruyue, CAO Yang, LI Cunyuan, ZHANG Yunfeng, HU Shengwei. Effects of circRNA-Zfp609 on Proliferation and Differentiation of C2C12 Myoblasts[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4535-4544.

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Effects of circRNA-Zfp609 on Proliferation and Differentiation of C2C12 Myoblasts

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