| CD151 is cancer gene of TM4 SF, which can promote the movement of cells and the invasion and metastasis of tumor. CD151 gene play its role of promoting the invasion and metastasis of tumor by promoting the angiogenesis and reascularization of tumor. It led to the destruction of shape and structure of the whole blood vessels after CD151 gene knockout. Wnt signaling pathway is a important signaling pathway which is closely related with the self-renewal and carcinogenic of cancer stem cells. LRG1 and LGR5 are both target genes of Wnt signaling pathways. LRG1 protein can promote the growth of tumor blood vessels, it plays a less role in the growth of normal blood vessels. LGR5 promote the angiogenesis, tumorigenesis and metastasis by promoting the expression of vascular endothelial growth factor(VEGF). Tumor cells in serum-free medium(SFM) can be a preliminary enrichment of tumorospheres which are rich in cancer stem cells. Cancer stem cells theory believe that cancer stem cells can be screened and separated accurately by identifying and using the surface markers of cancer stem cells, CD133 and CD44 are markers of colon cancer stem cells which is accepted by most scholars at present.Objective: This study use western blot method to investigate the correlation between CD151 and the expression of LRG1 and LGR5 protein which are related target genes in Wnt pathways in HT29 colon cancer cells and HT29 cells whose CD151 gene knockout. Screen the highly invasive CD133+ CD44+ HT29 and CD151- CD44+ CD133+ HT29 cell subsets by flow cytometry high-throughput colon cancer cell technology and detect the correlation between CD151 and the differential expression of LRG1 and LGR5 protein in tumor tissues which were formed by each cell subsets to explore the effect of the expression of CD151 on the invasion and metastasis of colon cancer.Methods:1 CD151- HT29 cells and CD151+ HT29 cells were detected by western blot method to detect the expression of CD151, LGR5 and LRG1 to explore the relationship between CD151 gene and LRG1, LGR5 in colon cancer cells.2 The expression of CD133+ CD44+ in CD151- HT29 cells and CD151+ HT29 cells which were cultured in serum free medium(SFM) and serum supplemented medium(SSM) was screened respectively by flow cytometry to screen the related subsets in cancer stem cells.3 Use cell proliferation assay and cell differentiation assay to identify the CD133+ CD44+ colorectal cancer stem cells isolated by fluorescece activated cell sorting.4 CD151- HT29 cells, CD151+ HT29 cells, CD151+ CD44+ CD133+ HT29 cells and CD151- CD44+ CD133+ HT29 cells which were cultured after flow cytometry sorting were grown in right axillary subcutaneous of 10 BALB/c-nu/nu nude mice to form tumor in nude mice, observed the tumor situation after 2 weeks.5 The tumor tissues which were formed by CD151- HT29 cells, CD151+ HT29 cells, CD151+ CD44+ CD133+ HT29 cells and CD151- CD44+ CD133+ HT29 cells were detected by western blot method to detect the expression of LGR5 and LRG1, to explore the relationship between CD151 gene and LRG1, LGR5 in colon cancer growing tumor.Results:1 Results of Western blot :(1) It has the expression of CD151 protein in HT29 cells, and doesn’t have it in HT29 cells whose CD151 gene were knockout, which tip the nonexistent of regulation role of CD151 gene in the cells,(2) The expression of LGR5 and LRG1 in CD151- HT29 cells was declined compared with CD151+ HT29 cells, the difference was statistically significant(P<0.05), show that lack of CD151 gene in HT29 human colon cancer cells significantly reduced the expression of LGR5 and LRG1 protein.2 Flow cytometry results showed that: the proportion of CD133+ CD44+ cells in CD151+ HT29 cells SSM group and SFM group were as follows: 0.3% and 1.4%, the difference was statistically significant(P<0.05); the proportion of CD133+ CD44+ cells in CD151- HT29 cells SSM group and SFM group were as follows: 2.7 % and 7.3 %, the difference was statistically significant(P<0.05), show that the proportion of CD133+ CD44+ in SFM HT29 cells was significantly higher than that in SSM HT29 cells.3 Cell proliferation assay showed: the number of HT29 cells increased with the increase in the number of days within 7 days i.e. OD value increased; the growth of CD133+ CD44+ CD151+ HT29 cells group is slower in the first two days than that of but HT29 cells group, but then increased linearly; the growth of CD133+ CD44+ CD151- HT29 cells group is relatively slow in the first four days, but increased in fifth days; the growth of CD151- HT29 cells group is slow within 7 days, the above growth trend of CD151- HT29 cells group, CD133+ CD44+ CD151+ HT29 cells group and CD133+ CD44+ CD151- HT29 cells group was significantly different from CD151+ HT29 cells group(P<0.01), show that compared to pre-election HT29 cells, the proliferation ability of CD133+ CD44+ CD151+ HT29 cells has improved significantly, the proliferation ability of CD151- HT29 cells has decreased. Cell differentiation assay showed: The cancer stem cell spheres formed in serum free medium can turn into adherent monolayer cancer cells in serum-containing medium, the morphology of cell don’t have obvious difference compared with the cultured HT29 cells before.4 Tumorigenicity assay in BALB/c-nu/nu mice: the nude mice in 4 group of CD151+ CD44+ CD133+ HT29 cells, CD151- CD44+ CD133+ HT29 cells, CD151+ HT29 cells and CD151- HT29 cells were all tumorigenic after 2 weeks, but the growth of growing tumor in CD151- CD44+ CD133+ HT29 cells group and CD151- HT29 cells group were both slow. The assay show that the colon cancer cells after screened have properties of stem cells, at the same time show that CD151 affected the growth of planting tumor.5 Results of western blot: The expression of LGR5 and LRG1 in the tumor tissues which were formed by CD151+ CD44+ CD133+ HT29 cells was significantly higher than that of the rest three groups, the expression of LGR5 and LRG1 in the tumor tissues which were formed by CD151- HT29 cells was significantly lower than that of the rest three groups, the difference was statistically significant(P<0.05).Conclusions:1 The lack of CD151 gene in HT29 cell can significantly decrease the expression of LGR5 and LRG1 protein.2 Serum free culture of HT29 cells is advantageous to the enrichment of CD44 and CD133 which are surface markers of cancer stem cells.3 The proliferation ability of HT29 colon cancer stem cells which were sorted by flow cytometry increased significantly, HT29 colon cancer stem cells can promote the invasion and metastasis of colon cancer.4 The expression of LGR5 and LRG1 in highly invasive colon cancer stem cells significantly enhanced, illustrate that the role of CD151 enhancing the invasion and metastasis of colon cancer may be mediated by Wnt pathways. |
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