Preliminary Study On MiR-302s Reprogramming Of Bama Pig Somatic Cells Into Induced Pluripotent Stem Cells

The miR-302 family is an embryonic stem cell(ESC)-specific miRNA.MiR-302s was found to enhance somatic cell reprogramming(SCR)efficiency as an enhancer,mouse and human induced pluripotent stem cells(iPSC)can also be obtained by using it alone.At present,miR-302s was proved to promote SCR of large livestock.There are few reports on the sequence and function of the large livestock of miR-302 family.To explore the function and mechanism of the miR-302 family in SCR of large livestock,the miR-302s sequence of buffalo was cloned and the eukaryotic expression vector was constructed.It was applied to reprogrammed Bama pig fibroblast(PFF)into iPSC.The cell transformation process and the difference of related gene expression in the process of inducing PFF into pluripotent stem cells by four transcription factors(OSKM)and miR-302s were investigated in detail.The results are as follows:1.Construction of buffalo miR-302s eukaryotic expression vectorThe lentiviral expression vector of buffalo miR-302s was constructed and analyzed by bioinformatics.The sequencing results showed that the buffalo miR-302 family sequence was obtained by PCR amplification,which is located in the intron region of the LARP7 gene on chromosome 7 of buffalo.The miR-302 family among members of the miR-302 family and between different species is highly conserved.A total of 255 major target genes of buffalo miR-302s were predicted.These target genes were mainly concentrated in 33 pathways,among which PGK signaling pathway and glucagon signal transduction and regulation of stem cell signaling pathway were the most significant.Bbu-miR-302s lentiviral particles can infect human,buffalo and pig somatic cells.2.Reprogramming of Bama Pig Fibroblasts into Induced Pluripotent Stem Cells by miR-302sBama pig somatic cells were infected with bbu-miR-302s virus.The results showed that miR-302s could reprogram Bama pig somatic cells into induced pluripotent stem cells(miR-302-piPS C).The obtained miR-302-piPSC had Alkaline Phosphatase activity,and RT-PCR results showed that miR-302-piPSC expressed endogenous OCT4 and SOX2 genes.Immunohistochemistry results showed that miR-302-piPSC expressed pluripotency markers OCT4,SOX2,FGF5 and STAT3.In vitro differentiation experiments showed that miR-302-piPSC could form embryoid bodies(EB)in vitro,and the expression of three germ layer marker genes could be detected.Compared with OSKM-mediated reprogramming,miR-302s-mediated reprogramming was inefficient and had a long cycle.The stem cell markers SSEA1,SSEA4,Tra-1-60 and Tra-1-81 were not detected in miR-302-piPSC,and there was no expression of endogenous gene NANOG.3.Study on Mesenchymal-epithelial Transformation of Bama Pig Fibroblast reprogrammed by miR-302sEpithelial-like morphological changes were observed in the process of miR-302s-mediated SCR.To explore whether mesenchymal-epithelial transformation(MET)occurred during miR-302s reprogramming,adhesion assay,Phalloidin staining,and detection of epithelial and mesenchymal cell molecular marker genes were performed in PFF after infection with miR-302s virus.The adhesion test results showed that the miR-302 group showed a more significant adhesion ability than the control group(P<0.01).The results of qRT-PCR showed that the expression of epithelial marker genes,including cell adhesion proteins(E-cadherin and Bves),tight junction protein Occludin,and cytokeratin(Krt18),increased significantly(P<0.05).Mesenchymal markers,including Vimentin,Fibronectin 1(FN1),protein collagen family Collal,and matrix metalloproteinase family(MMP14)were significantly down-regulated(P<0.05).The results of the phalloidin staining showed that over-expression of miR-302s destroyed the actin stress fibers of the cells compared to the control group.The above results indicate that MET occured during miR-302s-mediated reprogramming of pig fibroblasts.The results of qRT-PCR also showed that the mesenchymal cell-associated genes(Colllal,Vimentin,FN1,Snail,MMP14)increased first and then decreased with the increase of induction time.Epithelial cell-associated genes(E-cadherin,Bves,Krt18,Occludin)showed a trend of decreasing first and then increasing.It is indicated that during the induction of miR-302s,the somatic cells first pass through EMT,then pass MET,and then reach the pluripotent state.In the OSKM-mediated reprogramming process,the pluripotency state is directly passed through the MET.4.Differential expression analysis of related genes in miR-302s and OSKM-mediated reprogramming of Bama pig fibroblastsIn order to investigate the mechanism of miR-302s and OSKM in somatic cell reprogramming of Bama pig,RNA-seq was used to analyze and screen the differentially expressed gene(DEGs).The results of RNA-seq showed that,compared with the control group,there were 8887 DEGs in OSKM group and 4618 DEGs in miR-302s group.There were 3954 DEGs in both groups,accounting for 44.5%of DEGs in PFF vs OSKM group and 85.6%of DEGs in PFF vs miR-302 group.KEGG analysis showed that the DEGs specifically expressed by miR-302s was mainly enriched in ?-alanine metabolism,histidine metabolism,PI3K-Akt signaling pathway,extracellular matrix(ECM)-receptor interaction and glycolysis signaling pathway.The DEGs specifically expressed in OSKM was mainly enriched in oxidative phosphorylation signaling pathway,adhesion signaling pathway,Wnt signaling pathway and so on.Using the gene retrieval function of I-Sanger platform,the important target genes in reprogramming were queried and the expression trend map was drawn.Compared with the control group,the expression of epithelial marker gene closure protein(Occludin)and cytokeratin(Krt18,Krt 8)was up-regulated,the expression of mesenchymal cell marker genes Collal,MMP12,Snaill was down-regulated,and the expression of Vimentin,MMP14 and FN1 was up-regulated in miR-302 group.It is further suggested that EMT may exist in miR-302-mediated reprogramming.The expression levels of DNA methylase,methyl-CpG binding domain protein 2(MBD2)and histone deacetylase were down-regulated,indicating that miR-302 can affect SCR by regulating epigenetic.These results suggest that pig somatic cells can be reprogrammed into induced pluripotent stem cells by using miR-302 family alone.During miR-302s-mediated reprogramming,somatic cells undergo EMT,MET and finally form piPSC.MiR-302s may promote reprogramming through EMT-MET,Regulatory epigenetic,Glycolysis and PI3K signaling pathway.