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 p21by 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 Myomakerby 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.