Endothelial-derived GBP2 Regulates Differentiation Of Myogenic Cells

ObjectiveAge-related diseases,such as sarcopenia,are becoming a significant problem worldwide due to the aging of the global population.However,the clinical outcome of sarcopenia is still not promising.Therefore,to address this challenge,our group preformed single-cell sequencing of muscle,from which we found that GBP2 is associated with the occurrence and progression of sarcopenia and studied it in depth.Methods1.We first prepared an overexpression group lentivirus and a silencing group lentivirus of GBP2 and transfected mouse endothelial cells(C166).In each group of transfected cells,changes in the expression level of GBP2 were detected by RT-PCR method and Western-Blot method.Changes in proliferation,migration and tube-forming ability of each group of cells after lentivirus transfection were examined using Edu,cell scratch assay and tube-forming assay.2.D-Galactose induction was used to construct a sarcopenic cell model of C2C12 cells,and the appropriate drug intervention time and intervention concentration were screened using the CCK-8 method.Evaluation of D-Galactose-induced senescence in C2C12 cells by β-galactosidase staining and Western-blot method.Differences in the myogenic differentiation ability of the two groups of cells were detected using Jimsa staining and immunofluorescence staining methods.The difference in GBP2 expression between the two groups of cells was detected using Western-blot.3.After co-culture of endothelial cells and myogenic cells,the expression changes of myogenic differentiation markers MHC,MYOD1 and MYOG in each group of C2C12 cells were detected by RT-PCR method and Western-blot method,respectively.The relationship between GBP2 and STAT1 in C2C12 cells was verified by predicting the downstream proteins that may interact with GBP2;and using Western-Blot methods,immunofluorescence staining and immunoprecipitation experiments.Results1.The results of both RT-PCR and Western-blot experiments indicated that the expression level of GBP2 in C166 cells was steadily increased or decreased(P<0.05).The results of Edu assay,cell scratch assay and tube formation assay showed that cell lines overexpressing GBP2 inhibited cell proliferation,migration and tube formation compared to the control group,and conversely,cell lines silencing GBP2 increased proliferation,migration and tube formation(P<0.05).The results of ELISA experiments showed that the concentration of secreted GBP2 in the supernatant of cells in the overexpression GBP2 group was increased compared to the control group,while the concentration in the silent GBP2 group was decreased(P<0.05).2.The experimental results of CCK-8 showed that the OD value of C2C12 cells treated with 20 g/L of D-Gal was significantly lower compared to the control group(P<0.05).The results of both β-galactosidase staining and Western-blot experiments showed that senescence phenotypes were evident in the D-Gal group compared to the control group.The results of Jimsa staining and immunofluorescence staining showed that the ability of D-Gal to differentiate the constituent muscles was significantly decreased compared to the control group(P<0.05).The results of Western-blot assay showed that the expression of GBP2 was elevated in C2C12 cells of D-Gal group(P<0.05).3.The expression level of myogenic differentiation markers was significantly reduced in C2C12 cells co-cultured with overexpressed C166 compared with the control group by RT-PCR and Western-blot assay(P<0.05).The results of immunofluorescence staining showed that there was a co-localization relationship between GBP2 and STAT1 in C2C12 cells;immunoprecipitation experiments and Western-blot confirmed the reciprocal relationship between GBP2 and STAT1 in C2C12 cells.ConclusionsGBP2 affected the proliferation,migration and tube-forming ability of C166 endothelial cells;and a myasthenia gravis model of C2C12 cells was successfully constructed;finally,it was verified that endothelium-derived GBP2 entered into myogenic cells and affected their myogenic differentiation ability through STAT1 signaling pathway.