Regulating Mechanism Of CircSNX29 In Bovine Myoblasts Proliferation And Differentiation Via Wnt5a/Ca²⁺signaling Pathway

Myogenesis is a complex and precisely orchestrated process that is highly regulated by several non-coding RNAs and signal pathways.Circular RNAs（circRNAs）represent a novel subclass of endogenous non-coding RNAs that have been identified in multiple species and tissues and play a vital role in post-transcriptional regulation in eukaryotes,but the precise molecular mechanism of action remains largely unknown.Here we screened a candidate circRNA derived from the SNX29 gene,termed circSNX29 from our previous circRNAs sequencing data of bovine skeletal muscle,and further characterized its regulation and function during muscle development.The overexpression of circSNX29 facilitated myoblasts differentiation and inhibited cell proliferation.Computational analysis using RNAhybrid showed the potential for circSNX29 to sponge to miR-744 with nine potential binding sites.We tested this via a luciferase screening assay,and found that circSNX29directly interacted with miR-744 and down-regulation of miR-744 efficiently reversed the suppression of Wnt5a and CaMKIIδ.Importantly,through KEGG pathways enrichment analysis,Fluo-4,AM,Cell Permeant/Calciumion fluorescent probing and Western blotting assays,we found that ectopic Wnt5a and circSNX29 activated the non-canonical Wnt/Ca²⁺pathway by increasing activity of CaMKII and the levels of phosphorylated PKC.Taken together,the evidence generated by our study elucidates the regulatory mechanisms of circSNX29 to function as a sponge for miRNA-744 in bovine primary myoblasts.Our primary results are as follows:1.miR-744 regulates bovine primary myoblast proliferation and differentiation by targeting Wnt5aOur previous study found that several muscle specific or mature miRNAs,including miR-744,exhibit drastically changed expression in Qinchuan bovine muscle tissue.Variations in miRNA expression revealed significant up-regulation of miR-744.The effects of miR-744 on bovine primary myoblasts were studied by using EdU,MTT,CCK-8,RT-qPCR,Immunofluorescence Staining,and Western Blotting.We found that overexpression of miR-744 observably enhanced the mRNA and protein levels of muscle cell proliferation related-marker genes,such as CyclinD1,PCNA and CDK2,suggesting promoting cell proliferation.Inversely,overexpression of miR-744 dramatically down-regulated the expression levels of MyoG,MyoD and MyHC,suggesting that the miR-744 inhibited myoblast differentiation and the formation of myotubes.In addition,Luciferase reporter assay demonstrated that miR-744 promoted bovine primary myoblast proliferation and inhibited differentiation by regulating the post-transcriptional level of Wnt5a.2.circSNX29 serves as a competing endogenous miRNA-744 sponge to attenuate its inhibitory of Wnt5aTo identify specific circRNAs in bovine skeletal muscle,we randomly selected nine circRNAs from the circRNAs sequencing data,and showed that the expression of circRNA243 was much higher at the embryonic stage compared with the adult stage.As circRNA243 is transcribed from SNX29 gene,we termed it circSNX29.The online software RNAhybrid predicted that circSNX29 had 9 binding sites with miR-744,and Dual-luciferase reporter assay further revealed that circSNX29 did bind with miR-744,and overexpression of circSNX29 could significantly reduce the expression of miR-744.Results of EdU,MTT,CCK-8,RT-qPCR,Immunofluorescence Staining and Western Blot demonstrated that circSNX29 could observably promote myoblast differentiation and inhibit proliferation.These results were exactly opposite to effect of miR-744 overexpression on cell proliferation and differentiation.Meanwhile,circSNX29 enhanced the expression level of Wnt5a.Therefore,circSNX29 attenuates its regulatory effect on Wnt5a by adsorbing miR-744,thereby affecting bovine bovine primary myoblast proliferation and differentiation.Our study revealed a novel circSNX29 serves as an endogenous miR-744 sponge to activate Wnt5a/Ca²⁺pathway,resulting in inhibiting myoblast proliferation and promoting differentiation,which may be used as a potential target for regulation of myogenesis.