circRIPK2在鸡骨骼肌细胞增殖分化中的调控作用

目的 探索circRIPK2在鸡骨骼肌生长发育中的功能及作用机制。方法 依据反向剪切位点设计收敛、发散引物，并结合Sanger测序验证circRIPK2的环形结构。利用RT-PCR探究不同发育时期circRIPK2的表达水平。通过构建过表达载体，结合EdU、流式细胞术和RT-PCR，探究circRIPK2对鸡原代成肌细胞增殖分化的影响。结果 PCR电泳结果及Sanger测序证明circRIPK2环化位点真实存在。RT-PCR结果表明，和对照组相比，过表达circRIPK2后，对细胞增殖有抑制作用的标记基因p21的mRNA表达上调20%，对细胞增殖有促进作用的标记基因Cyclin B2、Cyclin D1、Cyclin D2和PCNA的mRNA表达分别下调39%、22%、29%和45%；肌分化标记基因MyHC、MYOG和Myomaker的mRNA表达分别上调了39%、56%和25%。EdU检测细胞增殖和流式细胞术检测细胞周期变化的结果表明，circRIPK2抑制细胞增殖进程。结论 circRIPK2可能通过抑制成肌细胞增殖、促进成肌细胞分化，从而影响鸡骨骼肌的生长发育过程。

Regulatory role of circRIPK2 in proliferation and differentiation of chicken primary myoblasts

Objective To explore the function and molecular mechanism of circRIPK2 on the growth of chicken skeletal muscle.Method The convergent and divergent primers were designed according to the back-splicing site, and the circular structure of circRIPK2 was verified by Sanger sequencing. RT-PCR was used to explore the expression level of circRIPK2 at different developmental stages. The circRIPK2 overexpression vector was constructed. The effect of circRIPK2 on the proliferation and differentiation of chicken primary myoblasts were detected using EdU, flow cytometry and RT-PCR technology.Result The electrophoresis of PCR product and Sanger sequencing proved the existence of circRIPK2. RT-PCR result showed that comparing with control, overexpression of circRIPK2 up-regulated the mRNA expression of cell cycle-inhibiting gene p21 by 20%, down-regulated the mRNA expressions of cell cycle-promoting genes Cyclin B2, Cyclin D1, Cyclin D2 and PCNA by 39%, 22%, 29% and 45% respectively, and up-regulated the mRNA expressions of differentiation marker genes MyHC, MYOG and Myomaker by 39%, 56% and 25% respectively. The EdU assay and flow cytometry analysis also indicated that circRIPK2 inhibited cell proliferation.Conclusion CircRIPK2 may inhibit myoblast proliferation and promote myoblast differentiation, thus affecting the growth and development of chicken skeletal muscle.