| Skeletal muscle satellite cells,which are located between the basal lamina and sarcolemma of myofibers,have the proliferation and differentiation potential as muscle-derived stem cells.The growth and development of skeletal muscle after birth were regulated by the expression of multiple genes,signaling pathways and network.The Notch signaling pathway plays an important role in the proliferation and differentiation of porcine skeletal muscle satellite cells(PSCs),but the key genes involved is still need to be found.In this study,the Illumina Hi Seq?2500 technology was adopted to detect the differentially expressed m RNA profiles between PSCs stably transfected with Notch1 Intracellular Domain(NICD)and the cells not transfected.Biological analysis was conducted to screen out several key candidate genes which could regulate the the porcine skeletal muscle satellite cells after proliferation and differentiation of porcine skeletal muscle satellite cell,including two genes,HEYL and RPL15,were selected to explore the roles in the regulation of the cells to provide the molecular theorical basis for the skeletal muscle growth and development regulation.1.RNA samples were prepared from PSCs stably transfected with NICD and PSCs that were not transfected,respectively,in proliferation stage and in differentiation stage,for the gene expression profile sequencing,and the results are as follows.Compared with Control group,there were 160 differentially expressed genes in NICD Group in proliferation stage,among which 93 genes were up-regulated and 67 genes were down-regulated.Compared with Control group,there were 13 differentially expressed genes in NICD group in differentiation stage,among which 7 genes were up-regulated and 6 genes were down-regulated.Compared with NICD group in differentiation stage,there were 1217 differentially expressed genes in NICD group in proliferation stage,among which 565 genes were up-regulated and 652 genes were down-regulated.Compared with Control group in differentiation stage,there were 1021 differentially expressed genes in Control group in proliferation stage,among which 486 genes were up-regulated and 535 genes were down-regulated.2.HEYL is a downstream target gene of Notch1.A luciferase reporter vector inserted with the HEYL gene promoter was constructed and the gene promoter activity was detected in different groups after transfection of HEK293 cells.Combined with the current research and the bioinformatics analysis,a binding site for NICD transcriptional activation was found on the HEYL gene promoter.The experimental result of Ch IP showed that the sites from-2309 to-2303 of HEYL gene was pulled down by the NICD antibody,confirming the existence of binding sites.PSCs were isolated from 1 day-aged landrace pig and cultured in vitro,and over-expression of HEYL and RPL15 were conducted,respectively,to study the effect on the proliferation and differentiation of PSCs and the regulation mechanism.3.HEYL promotes the proliferation of PSCs.Over-expression of HEYL gene raised the percentage of the proliferating PSCs detected by MTT assays and Ed U proliferation assays,thus increased the total number of cells.The expression of Pax7,CCNB1,CCND1,CCND2 was significantly increased after over-expression of HEYL,while the expression of P21 was significantly decreased detected by q RCR and Western blot,consequently promoting more cells entering into cell cycle and improving the cells proliferation.However,there was no significant changes in the expression of Myo D after over-expression of HEY gene.4.HEYL gene represses PSCs differentiation.PSCs were induced differentiation with 2% horse serum medium after over-expression of HEYL.The results showed that the expression of Myogenin(Myo G),Myosin and My HC gene expression were significantly decreased in HEYLover-expression group than the control group.These results suggested that HEYL gene repressed PSCs differentiation probably by inhibiting the expression of Myo G and My HC.5.RPL15 gene promotes the proliferation of PSCs.Over-expression of the RPL15 gene accelerated the proliferation of PSCs by MTT assays and Ed U proliferation assays.However,no significant difference was observed in the expression of P21,CCNB1 and CCND1 after over-expression of RPL15,suggesting that RPL15 may regulate PSCs proliferation by other factors.6.RPL15 gene represses PSCs differentiation.PSCs were induced differentiation with 2% horse serum media after over-expression of RPL15.The results showed that Myo G,Myosin and My HC expression was significantly decreased in RPL15-over-expression group than the control group.These results suggested that RPL15 represses PSCs differentiation probably by inhibiting the expression of Myo G and My HC.In addition,previous study has shown that mi R-22 may play an essential role in skeletal myogenesis,thus,the regulation of mi R-22 on the proliferation and differentiation of PSCs was explored in this study.7.The molecular mechanism of mi R-22 in skeletal muscle development was identified.First,a gradually increase in the expression of mi R-22 is observed during PSCs differentiation.After over-expression of mi R-22,the expression of CCNB1 and CCND1 was significantly decreased,while the expression of P21 was significantly increased by q RCR and Western blot.After inhibition of mi R-22,the expression of CCNB1 and CCND1 was significantly increased,while the expression of P21 was significantly decreased.The results aboved indicated that mi R-22 prevented PSCs from entering into cell cycles,eventually inhibiting PSCs proliferation by down-regulating the expression of CCNB1 and CCND1,and up-regulating the expression of P21.8.mi R-22 promotes PSCsdifferentiation.After over-expression of mi R-22,the expression of Myo G and My HC genes was significantly higher than that in the control group by q RT-RCR and Western blot.After interference with mi R-22,Myo G and My HC expression was significantly lower than that in the control group.These results indicated that mi R-22 could promote PSCs differentiation by improving the expression of Myo G and My HC.In this study,the candidate genes that may be involved in the regulation of Notch signaling pathway were selected,among which the regulation of HEYL and RPL15 on PSCs proliferation and differentiation was confirmed.Moreover,a micro RNA playing an important role in the proliferation and differentiation of PSCs was identified. |
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