Circular RNA ACTA1 Regulate Bovine Myoblasts Development Through The MAP3K11/MAP2K7/JNK Pathway

Circular RNAs(circRNAs)are a special class of noncoding RNA molecules,which can regulate many different biological processes.Skeletal muscle is an integral part to animal carcass.The growth and development level of skeletal myocyte will directly affect the meat yield and meat quality.The generation of skeletal myocyte is a complex process,which is mainly regulated by myogenic regulatory factors and various non-coding RNAs.Qinchuan cattle is a representative local cattle breed in China,which has excellent meat value.In recent years,a large number of studies have shown that circRNAs,is a new regulatory factor in the process of muscle development,can regulate the growth and development of skeletal myocyte.Therefore,it is important to explore the regulatory mechanism of circRNAs in the growth and development of skeletal myocyte in Qinchuan cattle to improve yield and quality of beef.Through the analysis of sequencing data in the early stage of the laboratory,a circRNA with differential expression fetal cattle and adult cattle in Qinchuan was successfully screened,and it was named circACTA1 according to its maternal gene.In this study,circACTA1 was selected as the research object to explore the function and mechanism of circACTA1 in Qinchuan cattle myoblasts by constructing the overexpression vector of circACTA1 and synthesizing the interference fragment of circACTA1.The main results are as follows:1.Identification and function of circACTA1 in bovine myoblastscircACTA1 comes from the second and third exons of ACTA1 gene.The authenticity of circACTA1 is verified by forward and reverse primer amplification,RNase R treatment and Actinomycin D treatment.The results show that circACTA1 is real.The analysis of expression characteristics showed that circACTA1 was expressed in different tissues of both adult and fetal cattle,and the level of circACTA1 expression was remarkably higher in adult cattle muscle than in fetal.The functional verification of circACTA1 showed that it could inhibit the multiplication of bovine myocyte and promote the apoptosis and cytodifferentiation of bovine myocyte.Nucleocytoplasmic isolation test showed that circACTA1 was mainly located in the cytoplasm.2.circACTA1 regulates the proliferation,apoptosis and differentiation of bovine myoblasts by adsorbing mir-199a-5p and mir-433RNAhybrid predicted that circACTA1 could be adsorbed by miR-199a-5p and miR-433.Double Luciferase Report test and RIP test showed that circACTA1 could be used as a molecular sponge for miR-199a-5p and miR-433.Functional tests showed that miR-199a-5p and miR-433 could promote the proliferation of bovine myoblasts and inhibit the apoptosis and differentiation of bovine myoblasts,while circACTA1 could reverse these effects.3.circACTA1 regulates MAP3K11/MAP2K7/JNK pathway by adsorbing miR-199a-5p and miR-433Targetscan 7.2 predicted that MAP3K11 and MAPK 8 may be the direct targets of miR-199a-5p and miR-433.The Double Luciferase Report test showed that miR-199a-5p and miR-433 could directly bind to MAP3K11 and MAPK 8,respectively.Functional verification showed that MAP3K11 and MAPK 8 could inhibit the proliferation of bovine myoblasts and promote the apoptosis and differentiation of bovine myoblasts.The activation of circACTA1 on MAP3K11/MAP2K7/JNK pathway was verified by RT-q PCR and WB test.The results showed that overexpression and interference of circACTA1 could activate and inhibit the expression of related genes on MAP3K11/MAP2K7/JNK pathway.4.Study on the effect of circACTA1 on the repair of muscle injury in miceClustal Omega analysis showed that circACTA1 had a high similarity in other species.The agarose gel test showed that circACTA1 existed in sheep and mice.A mouse muscle injury repair model was constructed to verify the function of circACTA1 in muscle injury repair in mice.The results showed that circACTA1 could enhance the repair effect after muscle injury in mice.In conclusion,circACTA1 activates MAP3K11/MAP2K7/JNK pathway by adsorbing miR-199a-5p and miR-433,thereby inhibiting the proliferation of bovine myocyte and promoting the apoptosis and differentiation of bovine myocyte,which further improved the molecular regulation mechanism in the process of myogenesis and provided new insights for beef cattle muscle development and breeding.