The Role Of Kirrel2 And KirrelA In Differentiation And Fusion Of Mouse Skeletal Myoblasts

Myogenesis occurs in multiple stages of life in many organisms,including embryonic development and growth and regeneration of adult animals.The key event in the process of muscle formation is the fusion of myoblasts to form new multinucleated muscle fibers or fusion with existing muscle fibers,thereby further increasing the number of muscle cores.Cell membrane fusion is a complex process that requires recognition,adhesion,cell signaling,cytoskeletal changes,and membrane rearrangement.Through research on Drosophila embryos,it was found that the transmembrane proteins Rst / Kirre and Sns expressed by founder cells(FCs)and fusion competent myoblasts(FCMs)promote the fusion of the two cells.The vertebrate orthologs of Rst / Kirre and Sns are Kirrel and Nephrin.Previous studies have shown that Nephrin plays a key role in the fusion process of mouse skeletal muscle myoblasts.The Kirrel gene family includes Kirrel1,Kirrel2,and Kirrel3.Previous experiments have shown that Kirrel A(a Kirrel1 subtybe)plays a role in the myoblast fusion process.Related studies of Kirrel2 have mainly focused on the kidneys and brain,but have not been studied in skeletal muscle.Therefore,this study will explore the effects of Kirrel2 on the differentiation and fusion of mouse skeletal muscle myoblasts and mechanisms of knocking down of Kirrel A to inhibit myoblast fusion was further investigated.Objective1.The role of Kirrel2 in the differentiation and fusion process of mouse skeletal muscle myoblasts.2.The mechanism of knocking down of Kirrel A in inhibition of myoblast differentiation and fusion.Methods1.Semi-quantitative RT-PCR and real-time quantitative RT-qPCR were used to detect Kirrel2 expression level during myoblasts differentiation and fusion and muscle regeneration after skeletal muscle injury.2.Construct Kirrel2 overexpression plasmid,overexpress Kirrel2 in mouse myoblast cell line C2C12,and compare the differentiation and fusion of myoblasts with the control group and calculate the fusion index.Immunofluorescence was used to detect the expression level of functional genes.3.Using existing Kirrel A-and Control-sh RNA C2C12 cell lines,compare the differentiation and fusion of myoblasts and calculate the fusion index.Western Blot and immunofluorescence were used to detect the expression level of functional genes.4.MTT and flow cytometry were used to detect the proliferation of Kirrel Aand Control-shRNA C2C12 cell lines.5.Use cell scratch assay to analyze the migration of Kirrel A-,Control-sh RNA C2C12 cell lines.Results1.The results of semi-quantitative RT-PCR and real-time quantitative RT-q PCR showed that Kirrel2 m RNA expression level first increased and then decreased during mouse myoblasts differentiation and fusion,and muscle repair after glycerin injury.2.The results of immunofluorescence showed that the fusion index of the C2C12 cell over-expressing Kirrel2 had no significant difference compared with the control group,and the size and shape of myotubes in two groups were similar.3.The results of immunofluorescence showed that the fusion index of Kirrel A-sh RNA was lower than that of Control-sh RNA on the second and fifth day of C2C12 differentiation.Western Blot results showed that the level of myosin protein in Kirrel A-sh RNA cells was lower than that of Control-sh RNA cells during C2C12 differentiation and fusion,but there was no significant difference in myogenin expression level.4.Both MTT and flow cytometry analysis showed that the proliferation rate of Kirrel A-sh RNA was significantly lower than that of Control-sh RNA.5.Cell mobility assay did not show any significant difference between Kirrel A-sh RNA and Control-sh RNA groups.Conclusions1.Kirrel2 expresses at very low in mouse myoblast,but its expression level increases during myoblast diferentiation and fusion,as well as in muscle development and reconstruction after injury.2.Overexpression of Kirrel2 has no effect on C2C12 differentiation and fusion.3.Knockdown of Kirrel A inhibits C2C12 proliferation and fusion.4.Knocking of Kirrel A has no effect on C2C12 migration during differentiation and fusion process.