Preliminary Study On The Regulation Of Proliferation And Differentiation Of Buffalo Myoblasts By CircCOPS8

Buffalo is mainly distributed in the south and is one of the characteristic species in Guangxi.Studies have shown that buffalo contains certain polyunsaturated fatty acids and conjugated linoleic acid,which is beneficial to human blood lipid health after long-term consumption.Circ RNA is one kind of non-coding RNA,which can regulate the growth and development of skeletal muscle in many ways,but there are relatively few studies on the regulation of buffalo muscle development by circ RNAs.Based on the previous RNA-seq data of our research group,we screened out the differentially expressed circ RNAs in the longissimus dorsi muscle of cattle and buffalo.We finally screened out the differentially expressed circ COPS8 as the research object,and verified the function of circ COPS8 on the proliferation and differentiation of buffalo myoblasts by constructing a vector.Through RNA-seq sequencing of buffalo myoblasts overexpressing circ COPS8,combined with q PCR and other experiments,we further analyzed the main pathways and possible mechanisms of circ COPS8 regulating buffalo myoblasts.The main results are as follows:1.The authenticity of circ COPS8 was verified by RNase digestion and q PCR,and the overexpression vector of circ COPS8 was constructed.The results of Ed U(P < 0.01)and CCK8(P< 0.01)showed that circ COPS8 significantly inhibited cell proliferation.The expression of proliferation marker genes was detected by q PCR and WB,and the results showed that the expression of PCNA and Cyclin D1 decreased significantly.The cell cycle detected by flow cytometry showed that circ COPS8 significantly increased the proportion of G0/G1 phase cells of buffalo myoblasts.2.To explore the effect of circ COPS8 on the proliferation of buffalo myoblasts.WB and q PCR detection results showed that the overexpression of circ COPS8 up-regulated the expression of Myo D and Myo G.The results of cell immunofluorescence test showed that the number of myotubes in buffalo myoblasts was higher than that in the control group after overexpression of circ COPS8.3.The model of muscle injury in mice was established to verify the effect of circ COPS8 on the repair of muscle injury in mice.The results showed that circ COPS8 inhibited the repair of muscle injury in mice.The marker genes of cell proliferation and differentiation were detected by WB and q PCR.The results showed that circ COPS8 could promote the cell differentiation of mouse muscle and inhibit cell proliferation.The effect of circ COPS8 on apoptosis marker genes of buffalo myoblasts and mouse myoblasts was further detected by q PCR.The results showed that circ COPS8 significantly promoted the apoptosis of buffalo myoblasts and mouse myoblasts.4.After RNA-seq sequencing and q PCR verification of buffalo myoblasts overexpressing circ COPS8,the regulatory network of differential genes was constructed.The results showed that circ COPS8 could activate tumor suppressor genes and inhibit cell proliferation.RIP-q PCR results showed that circ COPS8 could bind with IGF2BP3 to inhibit cell proliferation(P< 0.01).To sum up,the functional verification and mechanism discussion of circ COPS8 were carried out in this paper,which provided a theoretical basis for the research on the related functions and mechanisms of circ RNAs regulating buffalo muscle development.