Regulation Of Overexpression Of N1ICD On Proliferation And Differentiation Of Porcine Skeletal Muscle Satellite Cells Based On Expression And Small RNA Sequencing

Pig can not only be used as important meat-producing animal in livestock production,but also as a good model be used to researches.Muscle satellite cells are musclederived stem,which are located between the basal lamina and sarcolemma of myofibers.A series of myogenesises of muscle satellite cells,including its activation,proliferation,differentiation,fused into myotubes and so on,have a decisive role in regulating skeletal muscle.Notch signaling pathway and miRNAs are involved in the regulation of myogenesis of satellite cells,but for the interaction between them,which participates in the co-regulation of porcine skeletal satellite cells(PSCs)development,has not been reported.Notch1 gene regulate Notch signaling pathway mainly though N1ICD(Notch1 intracellular domain).Therefore,the model of stably transfected N1 ICD in PSCs had been established by our research group before.And,the cell samples from this model were collected on the proliferation and differentiation(seventh day),and carried out by gene expression profile and small RNA sequencing,which use to study the regulation of Notch1 in PSCs.It this study,we integrated the data of genes and mi RNAs profile to analyze differential expression miRNAs and genes,and constructed miRNA-mRNA regulatory network by bioinformatics.This is the first time that thoroughly revealed the regulatory network on proliferation and differentiation of PSCs by Notch signaling pathways.The results of this study are as follows: 1?Overexpression of N1 ICD in PSCs proliferation295 differentially expressed genes(DEGs)were obtained by gene sequencing data of PN1 ICD vs P-Control,including HEYL,HEY1,HES4 and JAG1 which are important genes in Notch signaling pathway,were also significantly up-regulated.Subsequently,the results of GO and KEGG enrichment analysis shown that the up-regulated DEGs mainly enriched in the intracellular biological process,such as cell cycle and cell proliferation,while the down-regulated DEGs mainly enriched in the extracellular biological process,such as cell adhesion and protein degradation.In addition,STRING database analysis demonstrated 11 up-regulated DEGs as core hub genes,including AURKA,AURKB,PLK1,CDC20,CCNB1,CDC25 B,CCNB3,TGB1,ESPL1 and CCNA2,which are involved in cell cycle process;and three down-regulated DEGs also as hub genes,including THBS2,ACTC1 and FGF2(the most hub genes),which associated with cell adhesion and migration.Thus,overexpression of N1 ICD in proliferative PSCs,promotes cell cycle regulation,but inhibits biological processes related to extracellular matrix.A total of 48 differentially expressed miRNAs(DEMs)were identified by analyzing the small RNA sequencing data of the P-N1 ICD vs P-Control samples.Among all the DEMs,the quantity of known DEMs and novel DEMs were 44 and 4,respectively,and musclespecific miRNAs(miR-206 and miR-133a-5p)were significantly down-regulated.Thus,known DEMs seem to play a major regulatory role than novel DEMs.Then,the target genes of known DEMs were predicted by miRWalk 2.0 database,and analyzed by KEGG.The results found that known DEMs are mainly enriched in pathways related to proliferation,such as MAPK?WNT and Insulin signaling pathways.Since DEMs and DEGs are mainly involved in regulating the proliferation of PSCs,according to the principle of miRNA negatively regulating genes,correlation analysis of known DEMs and DEGs were carried out,and the miRNA-mRNA interaction network were constructed,which want to obtain improtant miRNA-mRNA pairs in the regulation of PSCs proliferation.In down mi RNAs-up mRNAs network,ssc-miR-214,ssc-miR-423-5p,sscmiR-149 and ssc-miR-1343 are hub miRNAs whose target genes including Notch signaling important genes(HES4?HEYL and JAG1);the hub genes MKI67 and WHSC1,which both target ssc-miR-214 and four miR-10 family(ssc-mi R-10?ssc-miR-10a-5p?ssc-miR-125 a and ssc-miR-125b).Thus,we infer thatthe most important miRNA-mRNA pair in the network is ssc-miR-214 targeting MIK67 and WHSC1.Similarly,there are four hub miRNAs(ssc-miR-27 a,ssc-miR-146a-5p,ssc-miR-369 and ssc-miR-221-3p)and three hub gene(RUNX1T1,FGF2 and ACTC1)in up miRNAs-down mRNAs network.Because FGF2 is the most hub genes in gene-gene network,so we deduce that ssc-miR-146a-5p and ssc-miR-221-3p targeting FGF2 is the most important miRNA-mRNA pair.The all above results indicate that overexpression of N1 ICD activates the Notch signaling pathway,which promotes the proliferation of skeletal muscle satellite cells by regulating the cell cycle.And the important genes,miRNAs and miRNA-mRNAs involved in this process remain further study.2?Overexpression of N1 ICD in PSCs differentiationA total of 13 DEGs were identified by DEG sequencing for D-N1 ICD vs D-Control samples,including seven pig novel genes and six genes(ACTG2,ADM2,CNN1,EGR1,HSPB7 and MAD2L1).After the analyzing by GO annotation and literature,only EGR1 promotes differentiation of skeletal muscle satellite cells.Thus,the down-regualtion of EGR1 may be in favour of suppressed PSCs differentiation.Similarly,13 known DEMs and 3 novel DEMs were identified by small RNA sequencing in PSCs differentiation.After analysis of literature,only ssc-miR-214,ssc-miR-146a-5p and ssc-miR-30b-5p participate in muscle satellite cell differentiation and inhibit myogenic differentiation.Therefore,the downregualtion of this there mi RNAs probably expedite PSCs differentiation,which is different from the result of EGR1.All in all,there are some DEMs and DEGs when over-expressed N1 ICD in PSCs differentiation,we speculated that Notch signaling pathway plays an important role in PSCs differentiation.3?Quantitative real-time PCR validationA total of 20 DEGs in gene expression profile were randomly selected for qRT-PCR validation,together with N1 ICD,and 18 DEGs have the same trend between two methods.Similarly,11 DEMs were selected from small RNA sequencing and used for qRT-PCR validation,and the qRT-PCR results were consistent with the sequencing results.Thus,the sequencing data are highly reliable.In conclusion,we investigated the effect of overexpression of N1 ICD on the proliferation and differentiation of PSCs from miRNA and gene levels.The accuracy of sequencing results was verified by qRT-PCR.The results of our sequencing data analysis provide valuable information for understanding the molecular mechanisms underlying Notch Signaling pathway in PSCs and skeletal muscle development.