Identification And Characterization Of LncRNA/mRNA In Porcine Induced Pluripotent Stem Cells

Porcine induced pluripotent stem cells?piPSCs?are of tremendous value in livestock production,mediccal and biological basics.However,low efficiency,poor quality,and uncertain security still limite wider application of iPSCs due to poor understanding of molecular mechanisms in maintenance and establishment of cellular pluripotency.Though long non-coding RNA?lncRNA?has been shown be playing key roles in differentiation of cells and development of life organism by regulating gene expressions and so on,either in transcriptional or epigenetic level,so far reports on profiling of lncRNAs in porcine stem cells.Therefore,the present study aims to explore lncRNA and mRNA profiles in piPSCs in order to identify lncRNAs playing vital role in reprogramming.The results were as follows:?1?Through high-throughput sequencing,we found that there were 186,376,840 raw reads in porcine ear fibroblasts?pEFs?library,200,812,816 raw reads in porcine adipose-derived stem cells?pADSCs?library and 166,058,848 raw reads in piPSCs library,respectively.Ultimately,pEFs,p ADSCs and piPSCs library acquired 162,984,884,174,191,150 and 141,913,112 reads after removing the adaptor,low quality and rRNA sequence from raw datas,respectively.?2?To acquire the lncRNA expression profile,we then analyzed the lncRNA and mRNA transcriptome characteristics by bioinformatics.Firstly,comparing with the reference porcine genome,we have acquired 36617 known lncRNAs in the three libraries.Meanwhile,there were 32871 known mRNA coexpression among the three libraries,and 352,517 and 531 known mRNA were particularly expressed in pEFs,pADSCs and piPSCs,respectively.In addition,we also found 10506 known lncRNAs coexpressed in pEFs,pADSCs and piPSCs libraries.Besides,8099,8772 and 8285 known lncRNAs were expressed in the three cells,then 526,779 and 708 known lncRNAs specifically expressed among them.Finally,there were 4474 new lncRNAs in the 7815 no-coding potential transcripts,in which 3921 new lncRNAs were coexpressed among libraries.There were also 10,26 and 49 new lncRNAs specifally expressed in pEFs,pADSCs,piPSCs,respectively.?3?Differential expression,GO and KEGG analysis among piPSCs,pADSCs and pEFs.According to the principle of statistics and bioinformatics analysis,there were 648 genes differentially expressed in pEFs-pADSCs compare group in which 320 genes were up-regulation and 328 were down-regulation;In pEFs-piPSCs compare group,there were 3640 genes differentilly expressed between the two samples,in which 1825 genes were up-regulation and 1815 were down-regulation;Comparing the pADSCs with piPSCs,there were 2869 differentially expressing genes in which 1364 genes were up-regulation and 1505 were down-regulation.Meanwhile,there were 201 known lncRNAs defferentially expressed among the three kinds of cells,and 26,137,184 known lncRNAs were respectively expressed in pEFs-pADSCs,pADSCs-piPSCs,pEFs-piPSCs compare groups.Besides,there were also 5 new lncRNAs expressed amon the three libraries,including TCONS₀0082347,TCONS₀0023643,TCONS₀0090231,TCONS₀0064109 and TCONS₀0069653.Using KEGG analysis,we found that the differentially expressing genes were gathering 227,269 and 273 signaling pathways among those three compare groups,in which there were also 4926,8666,9279 GO functional groups enriched.In addition,the differential expressing genes were also enriched in the pluripotent signaling pathways and functional groups.?4?We then choosed 10 mRNA transcripts and 10 lncRNA transcripts to verify the sequencing datas.The results showed that the expression levels which detected through real time PCR were basically consistent with the sequencing analysis.So that our transcriptome datas were accuracy and reliability to represent the true different gene expression pattens among the three libraries.Then,we selected 24 lncRNAs which were differentially expressed among libraries for function prediction.There were 5 lncRNAs regulated by in cis control while 5 lncRNAs were regulated by in trans control.LOC102166377 and LOC102159188 target some genes which enriched in pluripotent pathways,but owing to down regulation in piPSCs,the two lncRNAs above may negative control pluripotency in stem cells.In coclusion,the study was the first to reveal both lncRNA and mRNA expression profiles in piPSCs by progressive high-throughput sequencing;Finally,we not only found a large number of lncRNA transcripts,but also predicted some lncRNAs which were closely related to establish and maintain pluripotency.We found 201 kinds of known lncRNA and 5 kinds of new lncRNA were significantly different among these three cells in the end.The gene IL-6,lncRNA LOC102166377 and lncRNA LOC102159188 may negatively control pluripotency by primary bioinformatics analysis.