Studies On The Oct4 Expression In Human Mesenchymal Stem Cells And Cancer Cell Lines And Its DNA Methylation

Objective: Study Oct4 expression in human mesenchymal stem cells(MSCs)and cancer cell lines and the role of DNA methylation in the regulation of Oct4 expression.Methods:1. UC-MSCs and BM-MSCs were isolated by tissue explants method and gradient centrifugation method, respectively;2. The multipotency of the isolated MSCs were confirmed by osteogenic and adipogenic differentiation;3. The MSCs specific surface molecule expression of the isolated MSCs were assayed by flow cytometry analysis;4. Real-Time PCR, ICC and FACS were performed to check the Oct4 expression in h IPS, UC-MSCs, BM-MSCs, DU145, PC-3, MCF-7, He La and 293T;5. DNA methylation specific PCR were done to analyze the methylation status of Oct4 regulatory regions in these cells.Results:1. UC-MSCs and BM-MSCs were isolated by tissue explants method and gradient centrifugation method, respectively;2. MSCs were induced to differentiate into osteogenic and adipogenic cells, confirming that the isolated MSCs are multipotent;3. Flow cytometry analysis showed that >95% of the isolated UC-MSCs could express the positive MSCs specific surface markers, but only <2% of the isolated UC-MSCs could express the negative surface markers, confirming that the isolated cells are MSCs;4. Real-Time PCR showed that all the checked cells could express Oct4 m RNA, and ICC and FACS showed that all the checked cells could express Oct4 protein;5. DNA methylation analysis demonstrated that in the 3 regulatory regions of Oct4 upstream, h IPS had a low DNA methylation, UC-MSCs and BM-MSCs had a medium level of methylation, and the other cells had a higher level of DNA methylation. But, in the region of Oct4 downstream(+37~+546), UC-MSCs had a medium level of DNA methylation while all the other cells including h IPS had a higher level of DNA methylation.Conclusions:1. The isolated MSCs were multipotent and expressed the MSCs-specific surface markers;2. Oct4 could be expressed in human MSCs and several cancer cell lines;3. DNA methylation could participate in the regulation of Oct4 gene expression in human MSCs and cancer cell lines. In addition, the regulatory mechanisms of Oct4 gene expression by DNA methylation are different between UC-MSCs and BM- MSCs.