AZA Treatment Of CEF Cells And Differential Expression Analysis And Function Prediction Of LncRNA/mRNA

Long non-coding RNA?lncRNA?is an important member of the non-coding RNA family and is a type of RNA molecule with a transcript longer than 200 nt that does not encode a protein.Studies have shown that lncRNA is a very important regulatory factor in the process of intracellular gene information transmission.It participates in much important biology and plays a crucial role in the maintenance of the pluripotency of embryonic stem cells?ESCs?.Induced pluripotent stem cells?iPSCs?are pluripotent stem cells derived from somatic cells induced by factors such as OCT4,SOX2,KLF4,and C-MYC?OSKM?,and are used in endangered animal protection and regenerative medicine.The study of iPSC is of great significance.However,its induction efficiency is very low.It has been reported that the demethylation reagent 5-Aza-2'-deoxycytidine?5-Aza-CdR,AZA,following called AZA?can replace one or more of the reprogramming factors?OSKM?to improve the efficiency of somatic cell reprogramming.However,how AZA affects the expression of IncRNAs and whether IncRNAs play a role in reprogramming are currently poorly understood.In this study,chicken embryo fibroblast?CEF?cells were treated with DNA methyltransferase inhibitor AZA.The methods of RNA-Seq and bioinformatics analysis were used to identify differentially expressed lncRNAs and pathway related to AZA-induced reprogramming.Then differentially expressed lncRNAs and mRNAs were verified by RT-qPCR.The main methods of research and results as follows:1.We selected CEF cells of treatment by AZA reagent and concentrate treatment time:CEF cells were treated with 5 ?M and 10 ?M AZA,and monitored the cells.And we found that 10 ?M AZA had a significant inhibitory effect on CEF.In the processing time,we consider the 48 h,96 h,and 120 h treatment time.After treatment with AZA?5 ?M?for 120 h,a large number of cells died;and after 96 h treatment,the NANOG gene was activated,and during this time,cells morphology changed.Therefore,the final selection of 5 ?M AZA treated 96 h for subsequent experiments.2.Analysis of IncRNAs after treatment of CEF cells by AZA:lncRNA sequencing analysis of AZA treated CEF cells for 96 h revealed 405 lncRNAs difference expression?include lncRNAs of 320 up-regulated,85 down-regulated?and 1029 mRNAs?include mRNAs of 646 up-regulated,383 down-regulated?of expressed significantly?P<0.05?,and the length of most IncRNAs were between 200-3000 nt.GO enrichment analysis showed that the genes of significantly upregulated IncRNAs are mainly involved in cell growth regulation?such as cell adhesion,migration,etc.?,while the downregulated IncRNAs are most closely related to the cellular immune function.The KEGG pathway analysis revealed that the WNT signaling pathway is activated and the MAPK is inhibited,which are closely related to the induction and reprogramming of iPSCs.3.Identification and expression analysis of a new lncRNA TCONS₀0370408:A novel lncRNA TCONS₀0370408 was screened from differentially expressed IncRNAs.The sequence was verified by PCR amplification and that sequencing was parallel with RNA-seq sequence.The fact shows that the sequencing results are authentic and reliable.In addition,RT-PCR results showed that TCONS₀0370408 was only expressed in CEF cells,but not in fully differentiated cells?HD11 and DF-1?,and AZA can induce its expression in CEF cells,This shows that the lncRNA may be related to the development and differentiation of embryos.In conclusion,in this study,transcriptome sequencing analysis of AZA-treated CEF cells was performed to obtain a transcriptional map of lncRNA/mRNA differentially expressed by AZA.And it has been identified the full-length of a novel IncRNA TCONS₀0370408 involved in the regulation of cell development and differentiation.The results provide abundant lncRNAs data for early embryonic development in poultry.Furthermore,it reveals the important role of lncRNAs in the regulation of iPSC reprogamming through activing WNT signaling pathway and inhabiting MAPK's and the close relationship between AZA-activated lncRNAs and iPSC induction efficiency.