Porcine EpCAM Regulates Cell Reprogramming And Expression Of Pluripotent Genes

Pig is not only an important economic animal,but also an ideal medical animal model.Derivation of bona fide porcine pluripotent stem cells is of great significance in animal breeding and regenerative medicine research.However,there is no report on the successful isolation of porcine ESCs up to date,strict problems still lie in the porcine iPS cells.Therefore,study of similarities and differences in signal transduction of porcine pluripotent genes towards mice and humans can help us understand the molecular regulation mechanism of porcine pluripotency.We found that epithelial cell adhesion molecule(EpCAM)was highly expressed in porcine iPSCs established home and abroad.In human and mouse,EpCAM can also be used as a marker of pluripotency for identification and screening of iPS and other pluripotent stem cells.In addition to use as a pluripotent marker,EpICD which was released from EpCAM during regulated intramembrane proteolysis(RIP)processes,can regulate several genes,by entering the nucleus.Here,we focused on the porcine EpCAM gene and protein.By profiling the expression of EpCAM in pig tissues and established pig iPS cells,a clear pattern of EpCAM expression in porcine pluripotency was depicted.Knockdown and overexpression of EpCAM in iPSCs and during cellular reprogramming to pluripotency revealed the relation between EpCAM and derived EpICD with other pluripotent genes.Finally,through further study on EpCAM protein regulation,mechanisms and pathways of EpCAM in pig pluripotent cells were clarified.1.Transcriptome profile and regulatory analysis of porcine EpCAM gene.In this study,we investigated the relation between EpCAM and porcine pluripotency by detecting the expression of EpCAM in piglet tissues,iPS cells,early embryonic development,and during cell reprogramming.By constructing the porcine EpCAM promoter reporter vector,we try to understand the expression of porcine EpCAM in iPS cells.The results showed that EpCAM expression was detectable in all tissues,and was higher in epithelial riched tissue.In the comparison of porcine iPS cells and porcine fibroblasts,the expression level of EpCAM in each iPS cell was higher than that of its precursor cell PEF,indicating that EpCAM was activated during reprogramming;EpCAM expression pattern was positively correlated with the key pluripotency gene,but negatively correlated with lineage specific genes.EpCAM expression was also correlated with MET gene.The expression pattern of pluripotent genes in early embryos showed that EpCAM is similar to that of key pluripotent genes.Additionally,OCT4 and SOX2 proteins have a down-regulate pattern of EpCAM promoter.KLF4,c-MYC,NANOG,LIN28,SALL4,ESRRB and other pluripotent transcription factors have an up-regulation effect on EpCAM promoter.2.Role of EpCAM during cellular reprogramming and in iPSCs.By successfully constructing EpCAM interfering shRNA vectors and EpCAM overexpression vectors,we found that knocking down of EpCAM reduced the AP positive colonies formation rate during PEF cells reprogramming,whereas overexpression of EpCAM significantly increased AP positive colonies formation rate.In DOX-iPS cells,knock down of EpCAM,resulted in reduction OCT4,SOX2,LIN28,SALL4 and ESRRB expression.While overexpression of EpCAM resulted in a significant up-regulation of these genes.Moreover,knockout of EpCAM in DOX-iPS makes the iPS colonies much looser,indicating that down-regulation of endogenous pluripotent genes caused by EpCAM silencing may lead to partial differentiation of iPS cells.3.EpCAM signalling pathways in pig.Through the study of the proteolytic process of porcine EpCAM,we found that by inhibiting the related proteases TACE and PS-2,the cleavage of EpCAM can be alleviated and translocation of EpICD into cytoplasm can be reduced.The detection of TACE and PS-2 expression levels in different cells of pigs also showed that there was a significant difference in m RNA levels between these cells,indicating that EpCAM may have different cleavage pattern in among cells to produce different abundances of EpICD to regulate its downstream genes.The addition of TACE and PS-2 inhibitors to DOX-iPS cells down-regulates the expression of EpCAM target genes,indicating that the transcriptional regulation of EpCAM is realized by EpICD produced by it’s cleavage.To demonstrate the role of EpICD,overexpression of EpICD in DOX-iPS cells under iPS culture conditions can promote the upregulation of the EpCAM target gene.Overexpression of EpICD in PEF cells can also improve the AP positive clones Formation rate.However,overexpression of EpICD in PEF under non-iPS cell culture conditions did not activate the promoter activity of EpCAM target genes.While once the GSK3 inhibitor CHIR99021 was added to the culture system,the EpCAM target gene promoter was activated.Because GSK3 has a effect on the degradation of beta-CATENIN,which indicates that EPCAM depends on the WNT / beta-CATENIN signaling pathway to perform transcriptional regulation of its target genes.