The Effect Of Circ-zfp609 On Proliferation And Differentiation Of Mouse Myoblast Cell

Circular RNA is noncoding RNA with closed circular structure.The existing studies reveal that circRNA plays an important role in biological process of eucaryote.Circular RNAs(circRNAs)have been identified in various tissues and cell types from human,monkey,porcine,and mouse.They can act as mi RNA sponges,regulators of transcription and translation,RBP sponges.However,there is few reports in skeletal muscle development.Base on existing literature and high-throughput sequencing results,we eventually selected circ-zfp609 as the subject to study.It was performed to identify the circular structure and expression profiles in different tissues and cell types.CCK-8 assay,RT-q PCR and Westerrn Blot were used to investigate the effect of circ-zfp609 on the proliferation and differentiation of differernt cell lines.Dual-Luciferase Reporter Assay,CCK-8 assay and RT-q PCR were employed to reveal the interaction between circ-zfp609,mi R-194-5p and downstream target gene BCLAF1.In addition,we explored the possibility that overexpression of mi R-194-5p sponge-acting circ-zfp609 increases the expression of mi RNA target gene BCLAF1 involved in the apoptosis.The main results of this study are as follows:(1)RNase R resistence and sequence analysis were used to reveal to the circular structure of circ-zfp609.It was confirmed as circular topology structure and the circularization sites were also consistent with the predicted results.The mapping analysis sequence of circ-zfp609 and genome based on circ Base showed a consistent results.(2)circ-zfp609 was highly conserved among different species,such as humans,mouse and cattle.The expression of circ-zfp609 in various tissues and cells showed that the circ-zfp609 expressed ubiquitously in mouse and Qinchuan cattle(fetal calves,calves)and various tissues(heart,liver,lung,kidney,stomach,intestine,mammary gland and leg muscle)and various cell types(C2C12,HC11,4T1,MCF-7 and MDA-MB-231),and the expression of circ-zfp609 in fetal bovine and bovine leg muscle were significantly different(P < 0.01),indicating that circ-zfp609 may regulate the muscle development.(3)CCK8 assay were performed to demonstrate the effect of siliencing circ-zfp609 on proliferation of various cell.The results showed that circ-zfp609 siRNA could promote the C2C12 growth and inhibit MCF-7 and MDA-MB-231 cells growth.Circ-zfp609 has little effect on Mammary epithelial cells(HC11 and 4T1).(4)Ctrl,scr siRNA,circ-zfp609 siRNA1 were respectively transfected into the C2C12 cells with Lipofectamine 2000 following the manufacturer's instructions.Cells were collected for RT-q PCR and Western Blot analysis against markers of proliferation and differentiation.The results showed that circ-zfp609 could inhibit the growth and differentiation.(5)Wild type and mutant type with circ-zfp609 luciferase reporter vectors were successfully constructed.The result of Dual-Luciferase reporters,RT-q PCR and CCK8 assay commonly demonstrated circ-zfp609 could act as mi R-194-5p sponges.(6)Circ-zfp609 significantly up-regulate m RNA expression of mi R-194-5p target gene BCLAF1.So we speculated that circ-zfp609 may mediatedly promote myoblast apoptosis through targeting BCLAF1.