Effect Of Id1/Id3 On Stemness Of Colon Cancer Cells And Its Mechanism

Objective Observed the effects of Id1/ Id3 on the stemness of colorectal cancer cells,and the mechanism of the effects on Wnt and SHH pathways that are important signaling pathways of colorectal cancer stem cells.Methods Lentivirus infection system was used to construct Id1/Id3 knockdown HCT116 cell lines and Id1/Id3 overexpressed normal intestinal epithelial cell lines NCM460,and cell lines overexpressing c-Myc in HCT116Sh-Id1 and HCT116Sh-Id3 cells.Real-time cell analysis system(RTCA),clone formation experiment and both flow cytometry analysis were used to detect cell proliferation ability.Western blot was used to detect the protein expression of related molecules.Serem-free spheres forming assay was performed to evaluate self-renewal capacity in vitro.The morphology of the cells was observed under microscopy and EMT changes were analyzed.The HCT116 cells were treated with the Wnt pathway inhibitor FH535 and the SHH pathway inhibitor HPI-1 for 48 h,respectively.And then western blot was used to detect the expression of related molecules.Results 1.The silencing of Id1 / Id3 gene in HCT116 cells significantly decreased the proliferative activity and clone formation ability(p < 0.05).The protein levels of PCNA,Survivin,cyclinD1,cyclinE,PI3 K,p-AKT,AKT were decreased by Id1/Id3 silence.In contrast,Id1/Id3 over-expression in NCM460 cells up-regulated the proliferation and clone formation ability(p < 0.05)and the protein levels of proliferation-associated proteins.2.Silenced Id1/Id3 in HCT116 cells the size and number of cell spheres formed in a serum-free suspension culture system also decreased significantly.Further analysis of stem cell makers showed that the expression levels of CD24,CD133,EPCAM,EZH2,Oct4,β-catenin and Lgr5 but not CD44 and CD166 were remarkably decreased in Id1/Id3 silenced HCT116 cells.However,NCM460 cells overexpressing Id1 / Id3 showed a significant increase in the ability of cell sphere formation and the expression of stem cell markers.3.The level Id1 / Id3-silenced cells were significantly down-regulated,and the protein levels of c-myc,cyclinD1 and survivin downstream of Wnt pathway were also significantly decreased.At the same time GLI1,GLI2 and Shh proteins of the SHH pathway were significantly down-regulated while PTCH2 and SUFU were significantly increased.The protein levels of c-Myc and CD133 in HCT116 cells treated with 5uM,10 uM Wnt pathway inhibitor FH535 and Shh pathway inhibitor HPI-1 decreased significantly,and the decrease of 10 uM concentration was more obvious.4.C-Myc overexpression in Id1-knockdown HCT116 cells significantly increased the ability of cell sphere formation and the expression of stemness related markers.Conclusion 1.Id1 and Id3 can activate the PI3K/AKT pathway by up-regulating the expression levels of survivin,PCNA,cyclinD1,and cyclinE,thereby promoting the proliferation of colorectal cancer cells.2.Id1/Id3 plays an important role in maintaining the stemness of colorectal cancer cells,including maintaining cell self-renewal capacity and tumorigenicity.3.Id1 / Id3 regulates stemness of colorectal cancer cells through Wnt and SHH pathways,and c-Myc may play an important role in this process.