| Skeletal muscle satellite cells(SCS)are a kind of muscle derived stem cells that exist between muscle cell membrane and matrix layer and have the potential of growth,reproduction and differentiation.They play an important role in the normal growth,regeneration and repair of muscle.The role of ubiquitin proteasome pathway in myogenic differentiation of bovine skeletal muscle satellite cells was explored in the laboratory.In the ubiquitin proteasome pathway,ubiquitin binding enzyme(E2)binds the activated ubiquitin and transfers the ubiquitin molecule to the substrate protein connected with ubiquitin ligase to be labeled by ubiquitin molecule.It is inferred that ubiquitin binding enzyme UBE2L3 plays a certain role in the development of bovine skeletal muscle satellite cells.1.In order to study the effect of ubiquitin binding enzyme UBE2L3 on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells(BSMSCs),the isolated BSMSCs were cultured in vitro and the myogenic differentiation model in vitro was established.To detect the changes of UBE2L3 mRNA and protein expression during the proliferation and myogenic differentiation of BSMSCs.The si-RNA of UBE2L3 was designed,UBE2L3 overexpression plasmid vector pcDNA-UBE2L3 and UBE2L3 ubiquitin binding enzyme active site mutation vector pcDNA-UBE2L3C86 were constructed,the expression of proliferation and differentiation markers of BSMSCs were detected by qRT-PCR and Western blot,and the effect of UBE2L3 on cell proliferation was detected by EdU staining,The effects of ubiquitin binding enzyme UBE2L3 on the proliferation and myogenic differentiation of BSMSCs were analyzed;Total cell protein was extracted to test whether UBE2L3 affected the level of ubiquitin protein in cells.The results showed that interference or overexpression of UBE2L3 had no effect on the proliferation of BSMSCs.After inducing the differentiation of BSMSCs,interfering with the expression of UBE2L3 will promote the myogenic differentiation of BSMSCs in vitro,while overexpression of UBE2L3 will inhibit the myogenic differentiation of BSMSCs in vitro.Western blot showed that the ubiquitinated protein level of BSMSCs would change after affecting the protein expression of UBE2L3.After further transfection of pcDNA-UBE2L3C86 vector,it was found that although the expression of UBE2L3 increased,it had no effect on the myogenic differentiation of BSMSCs.It shows that the effect of UBE2L3 on the myogenic differentiation of BSMSCs in vitro depends on its ubiquitin binding enzyme activity.The results laid a foundation for further exploring the regulatory mechanism of ubiquitination pathway in bovine myoblast differentiation.2.In order to further explore the regulatory mechanism of ubiquitin binding enzyme UBE2L3 on BSMSCs,the interaction between UBE2L3 and CDKN1B was detected by QRT PCR and Western blot.After transfection,the cells were cultured with CHX,and the total protein of BSMSCs was extracted at different time points for Western blot.It was found that changing the protein expression of UBE2L3 would affect the protein stability of CDKN1B.By using CDKN1B immunoprecipitation monoubiquitin and polyubiquitinated antibody for Western blot,it was found that the level of CDKN1B bound ubiquitin increased after overexpression of UBE2L3.It was further verified that UBE2L3 affected the expression of CDKN1B protein through ubiquitination pathway.After interfering with CDKN1B,it was found that it had no effect on the proliferation of BSMSCs.After BSMSCs were induced to differentiate,interference with CDKN1B inhibited the myogenic differentiation process of BSMSCs.In conclusion,ubiquitin binding enzyme UBE2L3 can regulate the myogenic differentiation of bovine skeletal muscle satellite cells.It was further found that UBE2L3 could regulate CDKN1B through ubiquitinated proteasome pathway,and then affect the myogenic differentiation of bovine skeletal muscle satellite cells.It will contribute to the study of the regulation mechanism of ubiquitination in bovine muscle development and myogenic differentiation. |
