Identification Of Functional Long Non-coding RNA During Porcine Pre-implantation Embryonic Development And IPSC Induction

Pig can serve as an ideal model for human diseases and development due to the similarity in their physiology and pathology.It is important to explore the regulation mechanisms about pig pre-implantation embryonic development,which are helpful to set up human diseases and development models,and to provide knowledge for pig breeding and its embryonic engineering.iPSCs appear to be a breakthrough in the field of stem cells,not only they are similar to embryonic stem cells and overcome the ethical problems,but also they could provide a reference for deriving the genuine porcine embryonic stem cells.However,the molecular mechanisms of pig pre-implantation embryonic development,somatic cell reprogramming and pluripotent maintenance of iPSCs are still not clear.In recent years,more evidence has been found that the IncRNA was involved in regulation of genes expression,thus IncRNA become the focus in the field of non-coding research.The length of IncRNA is usually longer than 200 nt,which play a role in modification of chromosomes and regulation of gene expression.Besides,the characteristics of spatial and temporal specific expression of IncRNA are more suitable for discovering its function during continuous embryonic development stages and reprogramming.To solve this problem,we performed bioinformatics analysis for pig pre-implantation embryos and iPSCs RNA-seq data,constructed the co-expression network of lncRNAs and protein coding genes and predicted the function of IncRNAs in embryonic development events and pluripotent maintenance.In order to study the functional IncRNA of embryos and iPSC,firstly,we performed stringent prediction pipeline and obtained the 226 high quality IncRNA genes（618 transcripts）.The distribution of length,count of exon and the sequence conservation of lncRNAs were similar to those in human and mouse,and novel IncRNAs also show a specific expression in embryonic stages.Furthermore,a co-expression regulatory network was constructed with IncRNAs and coding genes by the WGCNA package.25 modules which are related to the embryonic stages or cell types were found.In this paper we focused on the MElightcyan module involved in zygotic genome activation,the MEtan module involved in first lineage segregation and the MEblue module involved in pluripotency.In MElightcyan,GO and KEGG enrichment results show that genes of MElightcyan were related to synthesis,transport and degradation of RNA.We performed siRNA injection to knockdown the expression of XLOC₁26976 and this resulted in the reduction of blastocyst formation rate,whcih suggested that XLOC₁26976 was involved in regulating coding genes to affect zygotic genome activation;in MEtan,we found that XLOC₁55935 could serve as a potential marker for trophectoderm,which was validated by qPCR results,which consistent with RNA-seq expression;in MEblue,ChIP-seq data and synteney results indicated that XLOC 165754 could recruit transcription factors or epigenetic modifier and interact with neighboring genes by trans-acting and cis-acting to regulate reprogramming and maintain pluripotency.miRNA profiling of human-like and mouse-like porcine iPSCs were also analyzed in this study.The results indicated that the two types of cell were different in the signaling pathway of miRNA target genes.lncRNA could serve as miRNA target,and ssc-miR-4331 may interact with XLOC 139481,which specifically expressed in 8-cell stage embryo.It suggests that ssc-miR-4331 may participate in the regulation of embryonic development.In addition,the profiling of pig ICM and TE were also analyzed.Comparison of human and mouse counterpart data we found that there were differences in the signaling pathways related to pluripotency and we also screened key transcription factors that regulating the pluripotency.Besides,ICM specific endogenous retroviruses and its long terminal repeats may play a role in regulating the expression of lncRNAs.In summary,we identified functional IncRNA（XLOC₁26976,XLOC₁55935 and XLOC₁65754）during porcine pre-implantation embryos development and somatic reprogramming;besides,we also found the functional lncRNA interacts with miRNA and endogenous retroviruses that specifically expressed in blastocyst.This study provides new insights into discovering the molecular mechanism and regulatory network of porcine pre-implantation embryonic development;and it also provides the scientific evidence for further improving the quality of pig iPSCs;and also provides us a theoretical principle for isolating the porcine embryonic stem cells from inner cell mass.