Mechanism Of Circular RNA CDYL Regulating Myoblast Differentiation By Sponge MiR-665-5p

Background: Skeletal muscle in mammals has the potential to repair and regenerate itself in the event of injury or disease.Muscle repair is a multi-step process that involves muscle fibrosis,regeneration and remodeling.Myoblast differentiation is an important part of skeletal muscle repair,so it is important to study the underlying mechanism of myoblast differentiation.Studies have reported that microRNA,lncRNA and other non-coding RNAs play an important regulatory role in the differentiation of muscle cells.Recent studies have shown that Circular RNA(circRNA)is widely expressed in myoblast cells,suggesting that it may be involved in muscle growth and development.However,little research has been done on circRNA in myoblast differentiation,and the mechanism of action is still unclear.The purpose of this study is to provide theoretical basis for elucidating the mechanism of muscle regeneration under physiological and pathological conditions,and to provide basic support for the development of clinical intervention and drug targets for muscle atrophy.Methods: The expression of circCDYL and lineCDYL in C2C12 cells treated with RNase R or actinomycin D was detected by RT-q PCR.The subcellular localization of circCDYL was detected by fluorescence in situ hybridization(FISH).The effects of circCDYL on the proliferation of myoblasts were detected by RT-qPCR,EdU and CCK8.The effects of circCDYL on myoblast differentiation were detected by immunofluorescence and Western Blot.The miRNA adsorbed by circ CDYL sponge and its downstream target genes were found by bioinformatics analysis.The ceRNA mechanism of circCDYL was demonstrated by double luciferase reporting system,RTqPCR,Western Blot and immunofluorescence.The effects of lentivirus packaging on differentiation related genes in mice were detected by RT-qPCR and Western Blot after overexpression or knockdown of circCDYL in mice.The effect of circCDYL on repair of skeletal muscle injury was observed by immunofluorescence staining of tissue sections.Results: During the differentiation of C2C12 cells,the expression of circCDYL was gradually up-regulated,indicating that circCDYL plays an important role in the differentiation of myoblast cells.CircCDYL has the characteristics of circ RNAs and is mainly distributed in the cytoplasm.Overexpression of circCDYL can significantly inhibit the proliferation of myoblasts,and the expression of proliferation-related genes such as Cyclin D1 and PCNA is significantly down-regulated.Interference of circCDYL expression can promote proliferation.In addition,overexpression of circCDYL can promote differentiation of myoblasts,and the mRNA and protein levels of MyoD,MyoG and MyHC genes are significantly up-regulated.Interference with circCDYL can inhibit differentiation.Through bioinformatics analysis,it was screened and proved that circCDYL could regulate the proliferation and differentiation of myoblast cells by absorbing miR-665-5p by "sponge"-like manner.Further analysis of the miR-665-5p target identified the downstream target gene MASTR and fully demonstrated the regulatory network among the three elements of circcdyl-miR-665-5p-mastr.In vivo experiments in mice have demonstrated that circCDYL can regulate the expression of differentiation related genes in mice and promote the repair of skeletal muscle injury.Conclusion: CircCDYL inhibits downstream target gene MASTR by sponge adsorption of miR-665-5p,thereby inhibiting proliferation of myoblasts and promoting differentiation and repair of skeletal muscle damage.