Poly R(C) Binding Protein-1 Is A Negative Regulation To Cancer Stem Cells Activity In Prostate Cancer Cells

Part 1 Proteomics research on prostate cancer stem cells enrichment after TGF-?1 treatmentPurpose:To verify the role of TGF-?1 in the enrichment of prostate cancer stem cells.To explore the possible key proteins in the process of the enrichment of prostate cancer stem cells induced by TGF-?1.Methods: The stem cell markers CD44/CD24 of LNCa P and DU145 cells were identified after mock or TGF-?1 treated(7 days).Then CD44+/CD24-population was isolated from both prostate cell lines to detect stem cell marker CD133/?2?1Integrin,through fluorescent activated cell sorting.LNCa P cell lysates were obtained from the ±TGF-? cell population and proteomics profiling was performed by mass spectrometry.Results obtained from proteomics profiling were validated using Western blot in both LNCa P and DU145 cells.Result: TGF-?1 treatment caused significant enrichment of CD44+CD24-population in LNCa P and DU145 cells,which were also positive for CD133 and ?2?1Integrin.Mass spectrometry analysis of the enriched cell population revealed that sixty-three proteins were either up-or down-regulated greater than five folds,out of which the poly r(C)binding protein(PCBP)-1 was the most down-regulated(9.31 ± 0.05 folds).Through Western blot verification,while TGF-?1 induced LNCa P and DU145 cells enriched,PCBP-1 protein expression was gradually reduced.Conclusion: Under the induction of TGF-?1,CD44+/CD24-cells in LNCa P and DU145 cells were significantly enriched.And these cells were with high expression of stem cell markers CD133+/?2?1 Integrin+.While the stem cell enrichment was progressing,they were accompanied by a significant reduction in PCBP-1.Part 2 The regulation of PCBP-1 on cell proliferation and tumor growth of prostate cancer stem cellsPurpose:Validation of PCBP-1 reduction is an important condition for TGF-?1 induced prostate cancer stem cell enrichment.To study the effect of PCBP-1 on the proliferation and tumor formation ability of prostate cancer stem cells.Methods : PCBP-1 expression was up-regulated in LNCa P and DU145 cells by transfection and the expression of protein was verified by Western blot.Flow cytometry analysis of the expression of CD44,CD24 and CD133 in cells of PCBP-1 over expression group and control group after TGF-?1 induction.The effect of PCBP-1 on CD44,CD24 and CD133 transcription level(RNA)of stem cell markers were detected by q RT-PCR.In the over expression group and control group,the CD44+/CD24-cells were isolated,and the proliferation ability and tumorigenicity of these stem cells was verified by in vitro soft agar colony formation and in vivo xenograft assays.Result:The p CMV-AC-PCBP1-GFP plasmid was transfected into LNCa P and DU145 cells,and the stable clones were obtained by G418 screening.The high expression of PCBP-1 was confirmed by fluorescence microscopy and Western blot.The stem cells enrichment significantly decreased in these cells after induced by TGF-?1 compared with the control group.The q RT-PCR detection showed that the high expression of PCBP-1 was significantly correlated with the up regulation of CD24 and down regulation of CD133 at the RNA level(P<0.05).Soft agar colony formation assay and nude mice in vivo test confirmed that the control group of LNCa P and DU145 stem cell proliferation and tumor forming ability is very strong,and PCBP-1 significantly inhibited the characteristics of these stem cells.Conclusion: PCBP-1 played a negative role in the enrichment,proliferation and tumor forming ability of prostate cancer stem cells.