Identification And Function Analysis Of MicroRNAs In Porcine Pluripotent Stem Cells

Porcine pluripotent stem cells(pPSCs)have significant value in animal breeding,human medicine,and biological basic researches.However,to date,the understanding of molecular regulatory mechanisms engaging in establishment and maintaince of porcine cellular pluripotency is poor.MicroRNAs(miRNAs)are small,noncoding RNAs that serve as a crucial epigenetic modification in regulating eukaryotic cellular pluripotency and somatic cell reprogramming.Whether miRNAs play asimilar role in pPSCs has not been deeply explored.To investigate the role of miRNAs in porcine cellular pluripotency and reprogramming regulatory network,in the present study,we characterized and investigated the differential expression of miRNAs in porcine Ear Fibroblasts(pEFs),porcine adipose derived stem cells(pADSCs),porcine Partially reprogrammed iPSCs(pPiPSCs)and porcine Na?ve-like iPSCs(NpiPSCs)by using deep sequencing technology and quantitative real-time PCR(q-RT-PCR)analysis.Afterwards,in order to screen out the specific miRNAs in regulatory network of porcine cellular pluripotency and somatic cell reprogramming,the prediction of target gene function for differential miRNAs and analysis of the GO term and KEGG Pathway were conducted to further understand the potential role of miRNAs.The results were as follows:1.Using Solexa deep sequencing,this study finished the small RNA sequencing libraries of pEFs,pADSCs,pPiPSCs,NpiPSCs,and each sample in triplicate.After removing the reads of low quality and masking adaptor sequences,the clean reads are all accounts for approximately 95% of the high quality reads in every sequencing library.The majority of the small RNAs were 21-24 nt in size,and peaked at 22 nt.2.The different miRNAs pattern are shown in porcine cell types with different degrees of pluripotency or reprogramming.Compared with pEFs and pADSCs,the pluripotency associate miRNAs are expressed higher in NpiPSCs.For instance,miR-371-5p,miR-17/92 cluster,miR-106a/363 cluster and miR-182.The expressionof differentiation related miRNAs is opposite,such as let7 cluster and miR-145.miR-29 a and miR-21,two reprogramming suppressive miRNAs,express decreasingly in pADSCs,pPiPSCs and NpiPSCs.In pluripotency related miRNAs,miR-106a/363,miR-18 b,mi R-20 b,miR-18 a,miR-20 a were not significant differences in expression between pPiPSCs and NpiPSCs,but miR-371-5p and miR-182 were higher in NpiPSCs.Moreover,pADSCs as control,ssc-miR-219b-3p,ssc-mi R-7138-3p,ssc-miR-105-2,ssc-miR-1468,ssc-miR-136 and ssc-miR-96-5p express highly in NpiPSCs,and ssc-miR-31 expresses highly in pPiPSCs.pPiPSCs as control,ssc-miR-432-5p expresses highly in NpiPSCs.3.The target genes of candidate miRNAs were predicted by using mi Randa,targetscan and PITA software,after which the intersection was made.The GO and KEGG pathway analysis indicated that the target genes of candidate miRNAs in each library were enriched in signaling pathways of pluripotency,reprogramming and adipogenesis including the Pathway in cancer,JAK-STAT,WNT,Notch,metabolism and adipocytokine.Moreover,the target genes were enriched in molecular function related to regulation of pluripotency including regulation of transferase activity,nucleotide binding,protein binding,translational regulation and etc.In summary,the current study detected the profiling of miRNA in porcine cells with different degrees of pluripotency.miR-7138-3p,miR-432-5p and miR-31 may play a potential specific role in establishment and maintaince of procine celluar pluripotency.The present study enriched the understanding of epigenetic regulation and provided theoretical support for the application of porcine stem cells.