Regulating Mechanism Of CircTTN In Bovine Myoblasts Proliferation And Differentiation

Skeletal muscle is an essential tissue that is an important research object of meat production and meat quality.Myofibers in skeletal muscle are generated by fusion of multiple mononucleated myoblasts through a process known as myogenesis.Myoblasts undergo proliferation,differentiation,and eventually fuse into polynuclear muscle tubes,which are regulated by Myogenic Regulatory Factors(MRFs),including Myo D,Myf5,Myo G,and MRF4,and non-coding RNA(nc RNA).Circular RNA(circRNA)are ubiquitous endogenous RNA found in various organisms that can regulate gene expression in eukaryotes.In recent years,research on circRNA has received widespread attention.Relevant studies have shown that circRNA is involved in regulating myogenesis,and its importance cannot be ignored.Therefore,we analyzed previous circRNA sequencing data of bovine skeletal muscle tissue in our laboratory and found differential expression of circ Titin(circTTN)in fetal and adult bovine muscle tissue.Some experimental overexpression and knockdown techniques,RT-q PCR,cell immunofluorescence,Ed U,CCK-8,flow cytometry,dual-luciferase report analysis,RIP,and Western Blot were used to study the effect of circTTN on proliferation and differentiation of bovine myoblasts and its regulatory mechanism.It is expected to provide a new theoretical basis for the molecular breeding of Qinchuan cattle.The main results are as follows:1.Verification of authenticity for circTTN and its effect on the proliferation and differentiation of bovine myoblastscircTTN is derived from the TTN gene and has a length of 675 nt.The authenticity of circTTN was confirmed by RNase R digestion and Sanger sequencing.It was proved by RT-q PCR,Western Blot,flow cytometry,CCK-8,Ed U and immunofluorescence staining that overexpression of circTTN could promote proliferation and differentiation of bovine myoblasts,while knockdown of circTTN inhibited proliferation and differentiation of bovine myoblasts.2.circTTN regulates the proliferation and differentiation of bovine myoblasts as a molecular sponge of mi R-432The results of RNA-FISH and karytoplasmic separation showed that circTTN was mainly expressed in the cytoplasm,indicating that circTTN could act as a molecular sponge of mi RNA to regulate the proliferation and differentiation of bovine myoblasts.Using bioinformatics software RNAhybrid found that circTTN had potential binding sites for mi R-432,and the results of dual-luciferase gene reporting system and RIP showed that circTTN could interact with mi R-432.Via RT-q PCR,Western Blot,flow cytometry,CCK-8,Ed U and cell immunofluorescence assays,we found that overexpression of circTTN can reverse the inhibitory effect of mi R-432 on the proliferation and differentiation of bovine myoblasts3.circTTN regulates the IGF2/PI3K/AKT signaling pathway by adsorbing mi R-432Dual-luciferase gene reporting system and Western Blot assays proved that IGF2 was the direct target gene of mi R-432.Subsequently,using RT-q PCR,Western Blot and flow cytometry assays,it was found that knockdown of the IGF2 could inhibit the proliferation and differentiation of bovine myoblasts,indicating that IGF2 positively regulated the proliferation and differentiation of bovine myoblasts.The expression of IGF2/PI3K/AKT signaling pathway related genes after overexpression or knockdown of the circTTN was detected by RT-q PCR and Western Blot,and the results showed that circTTN could activate the IGF2/PI3K/AKT signaling pathway.Conclusively,our results showed that circTTN promoted proliferation and differentiation of bovine primary myoblasts via competitively combining with mi R-432 to activate the IGF2/PI3K/AKT signaling pathway,providing powerful help to reveal the complex molecular mechanism of myogenesis.At the same time,it also provides a new molecular marker for molecular breeding in the beef industry in China..