Effects of CHRNG Gene on Proliferation and Differentiation of Bovine Myoblasts and Its Mechanism
This study aimed to investigate the effect of CHRNG gene on the proliferation and dif-ferentiation of bovine myoblasts and its potential molecular pathways.In this study,adenovirus was used to overexpress and interfere with CHRNG gene in Qinchuan cattle myoblasts.This study isolated myoblasts from the longissimus dorsi and hind leg muscles of healthy 3-day-old Qinchuan cattle.Adenovirus overexpression and interference with CHRNG gene were used in Qinchuan cattle myoblasts,which were divided into interference group(sh-CHRNG),interfer-ence control group(sh-NC),overexpression group(OE-CHRNG),and overexpression control group(OE-NC),with 3 replicates in each group.CCK-8,EdU,qRT-PCR,Western blot,im-munofluorescence staining and other methods were used to detect the effects of interference and overexpression of CHRNG gene on the proliferation and differentiation of bovine myoblasts;Fur-ther screening of differential genes and enrichment of signaling pathways were performed through RNA-Seq.The results showed that overexpression and interference with CHRNG significantly downregulated the expression of cell cycle factors PCNA,CCNB1,and CCND2(P＜0.01),significantly upregulated the expression of CDKN1A(P＜0.01),reduced the number of proliferating cells(CCK-8,P＜0.01),and reduced the proportion of positive cells in the S repli-cation phase(EdU).Overexpression and interference of CHRNG on bovine myoblasts and in-duction of differentiation were observed by morphological observation and immunofluorescence staining on D2,D4,and D6.The results showed that interference and overexpression of CHRNG inhibited the differentiation of bovine myoblasts and myotube formation.The qRT-PCR and Western blot results showed that overexpression and interference with CHRNG downregu-lated the mRNA and protein levels of MYOD1,MYOG and MYH3 genes(P＜0.01).Through RNA-Seq sequencing analysis,it was found that the differentially expressed genes screened by overexpressing CHRNG were mainly enriched in endoplasmic reticulum protein processing,IL-17,thyroid hormone synthesis,PPAR,PI3K-Akt and other signaling pathways.Among the top 20 enriched pathways analyzed by KEGG,10 pathways were related to cell differentiation;The differentially expressed genes screened by CHRNG were mainly enriched in signaling path-ways such as axon guidance,MAPK,and PI3K-Akt.Among the top 20 enriched pathways ana-lyzed by KEGG,8 pathways were related to cell differentiation.The results suggested that over-expression and interference with CHRNG gene can inhibit the proliferation and differentiation of bovine myoblasts,and the pathways enriched by differential genes caused by overexpression and interference with CHRNG are mostly related to cell differentiation.
CHRNG geneproliferation and differentiationmyoblastRNA-Seq
万方数据
维普
