circRNA-Zfp609对C2C12成肌细胞增殖和分化的影响

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

【目的】试验旨在探究circRNA-Zfp609调节C2C12成肌细胞增殖和分化的潜在分子机制。【方法】利用RT-PCR和测序分析小鼠骨骼肌组织和C2C12成肌细胞中circRNA-Zfp609的表达,实时荧光定量PCR检测小鼠心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、骨骼肌组织及增殖12、24、36、48 h和分化0、1、3、5 d的C2C12成肌细胞中circRNA-Zfp609的相对表达量;实时荧光定量PCR检测分化0、1、3、5 d的C2C12成肌细胞中肌细胞生成素(MyoG)和肌球蛋白重链(MyHC)的相对表达量。用circRNA-Zfp609的干扰表达载体(siRNA)干扰细胞,通过CCK-8测定siRNA对C2C12成肌细胞增殖率的影响;用实时荧光定量PCR检测siRNA干扰对circRNA-Zfp609、MyoG和MyHC相对表达量的影响。通过TargetScan 7.0和miRDB软件预测circRNA-Zfp609上与肌肉分化相关的miRNA位点,将筛选的miRNAs的过表达载体转染HEK293T细胞,利用双荧光素酶报告试验验证circRNA-Zfp609与miRNA的互作关系。根据miRNAs对circRNA-Zfp609的互作,构建circRNA-Zfp609的野生型和突变型载体并转染HEK293T细胞,利用双荧光素酶报告试验验证circRNA-Zfp609对miRNA的靶向关系。【结果】PCR和测序结果表明,小鼠骨骼肌中可表达circRNA-Zfp609;circRNA-Zfp609在小鼠骨骼肌中表达水平最高,在其他组织中的表达量由高到低依次是肾脏、肺脏、心脏、肝脏、胃、脾脏和小肠。与12 h相比,在C2C12成肌细胞增殖的36和48 h circRNA-Zfp609的相对表达量显著增加(P<0.05);与分化第0天相比,在C2C12成肌细胞分化的第1、3和5天circRNA-Zfp609的相对表达量均显著增加(P<0.05),MyoG、MyHC的相对表达量均极显著增加(P<0.01)。与NC组相比,siRNA组C2C12成肌细胞的增殖率和circRNA-Zfp609相对表达量均极显著降低(P<0.01),MyoG和MyHC的相对表达量显著降低(P<0.05)。circRNA-Zfp609上有miR-150-5p、miR-327、miR-344g-3p和miR-615-5p 4种与肌肉分化相关的miRNAs。circRNA-Zfp609与miR-615-5p的吸附能力最强,具有靶向结合作用。circRNA-Zfp609可以作为分子海绵与调控肌肉分化相关的miR-615-5p相互作用。【结论】circRNA-Zfp609在小鼠的组织中广泛表达,在骨骼肌中表达水平最高;circRNA-Zfp609在C2C12成肌细胞增殖和分化的不同时期差异表达,circRNA-Zfp609上有4个与肌肉分化相关的miRNAs,其中miR-615-5p与circRNA-Zfp609具有靶向关系。本研究结果可为与家畜骨骼肌生长发育相关的研究提供参考。

【Objective】 The purpose of the experiment was to explore the potential molecular mechanism of circRNA-Zfp609 regulating the proliferation and differentiation of C2 C12 myoblasts.【Method】 The expression of circRNA-Zfp609 in mouse skeletal muscle tissues and C2 C12 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 C2 C12 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 C2 C12 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 C2 C12 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 HEK293 T 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 HEK293 T 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 C2 C12 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 C2 C12 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-150-5 p, miR-327,miR-344 g-3 p and miR-615-5 p) on circRNA-Zfp609 were related to muscle differentiation.circRNA-Zfp609 had the strongest adsorption capacity with miR-615-5 p, and had a targeted binding effect.circRNA-Zfp609 could act as a molecular sponge to interact with miR-615-5 p 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 C2 C12 myoblasts at different stages of proliferation and differentiation, circRNA-Zfp609 had 4 miRNAs related to muscle differentiation, and miR-615-5 p 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.