Analysis Of MiRNA-mRNA Regulatory Network In PiPSCs And The Mechanism Study Of Key MiRNAs In Pluripotent Regulation

Induced pluripotent stem cell(iPS cell)is a kind of pluripotent stem cells with the potential of self-renewal and multidirectional differentiation.Compared with embryonic stem cells,iPS cells not only have a wide range of cell sources,but also avoid the issues of ethics and immune rejection,therefore,they have the potential applications for the cell therapy and tissue regeneration.Currently,there are many defects that were reported in the established porcine iPS cells compared with mouse or human iPS cells.In-depth study of their molecular regulatory mechanism is needed to establish the na?ve porcine iPS cells and their culture conditions.In this study,mRNA-seq and miRNA-seq data were analyzed to screen and find out miRNAs that regulate the expression of pluripotent genes and manipulate the generation of porcine iPS cells.Thus,this study provides new ideas for optimizing the induction system of porcine iPS cells and obtaining a na?ve state porcine iPS cell line.Methods and results are as follows:1.Combined analysis of miRNAs and mRNAsWe performed miRNA sequencing on three piPS cell lines which generated from different culture conditions(piPS-L,piPS-F and piPS-LF),and obtained 165 specifically expressed miRNAs.According to miRNA target prediction as well as GO and KEGG analysis,it was found that some target mRNAs were involved in stem cell pluripotency regulation.Then,mRNA-seq data of these piPS cell lines were analyzed,and 1416 specific expressed mRNAs were obtained.Finally,we made conjoint analysis between miRNA target mRNAs and 1416 specifically expressed mRNAs,and obtained a miRNA-mRNA target map of piPS cells.There were 16 miRNAs and 13 target mRNAs in this map.2.Validation of the miRNAs target to LIN28 A gene and its regulatory role in piPSCs.According to the miRNA-mRNA target map,we made further study on the pluripotent gene LIN28 A.At first,the 3’UTR luciferase reporter system was established,and LIN28 A 3’UTR vector was transfected into 293 T cells with miR-370 and miR-31863,respectively.The luciferase assay was performed 36 hours after transfection.The results showed that only miR-370 could target to the 3’UTR region of LIN28 A.In our study,it was found that during the differentiation of piPSCs,the expression of LIN28 A was decreased,while the expression of miR-370 was significantly up-regulated.Next,the miR-370 lentivirus vector was constructed to infect piPSCs,it was found that miR-370 could inhibit endogenous LIN28 A expression,reduce other pluripotent genes expression,up-regulate differentiated genes expression,then lead to lower the alkaline phosphatase activity and self-renewal ability of piPS cells.LIN28 A rescue could significantly reverse these negative effects of miR-370.These results indicate that miR-370 can target LIN28 A to regulate pluripotent state of piPSCs.3.Prediction and verification of the miRNAs targeted OTX2 gene.By cluster analysis,we compared the expression profiles of pluripotent state-related genes in piPSCs which generated from different culture conditions.The results showed that the expression levels of OTX2 and ZIC3(Primed state genes)were five to ten-fold higher than those of NANOG and ESRRB(Na?ve state genes)in these three piPS cell lines.Then,six miRNAs which might target OTX2 gene were predicted through three miRNA prediction platforms.The luciferase assay results showed that two miRNAs of them(miR-1343 and miR-545)could specifically target and regulate the 3’UTR region of OTX2 gene.Finally,the miRNA lentivirus vectors were constructed to infect piPSCs,it was found that these two miRNAs could significantly inhibit the expression of OTX2 mRNA,thus inhibiting the expression of endogenous OTX2 protein.4.Regulation effects of miRNA-OTX2 on piPSCsIn this study,we found that miR-1343 and miR-545 could facilitate piPS cells proliferation,improve their alkaline phosphatase activity,up-regulate the expression of the pluripotent genes SOX2,NANOG and ESRRB,and repress the expression of OTX2 downstream genes FGF5 and OCT6.Within EB differentiation,it was found that miR-1343 and miR-545 could promote piPSCs differentiated into neuroectoderm.Furthermore,we found that OTX2 rescue could reverse all the positive effects of these two miRNAs.By promoter luciferase assay,we found that OTX2 could repress SOX2 and ESRRB promoter activity,and their promoter activity was also significantly activated in the piPSCs which treated with miR-1343 and miR-545,respectively.we also found that SOX2 could repress OTX2 promoter activity.Further study revealed that SOX2 and ESRRB could facilitate the expression of miR-1343,on the contrary,OTX2 could repress miR-1343 expression.But all the above transcription factors had no obvious effects on miR-545 expression.These results indicated that miR-1343-OTX2 axis participated in the regulation of self-renewal and differentiation of piPSCs.