Mast Cells Involved In Maintaining The Stemness Of Igr5~+Colonrectal Cancer Stem Cells And The Investigation Of Its Mechanism

Part I The expression of Lgr5+, RSPO-1, C-kit in human colorectal cancer and its clinical significanceObjectives:To investigate the expression of mast cells infiltrated in colorectal cancer, which expressed the RSPO-1protein. To discuss the clinical and pathological relationships between RSPO-1protein and infiltrating mast cells in human colorectal cancer.Methods:21cases of colorectal carcinoma, para-carcinoma and proximal surgical margin tissues which were adjacent cancer tiusses were obtained from patients between March2012and April2012. The expression of RSPO-1and C-kit protein was detected in different tiusses with immunohistochemical staining, immunofluorescence and Western blot, with real-time quantitative polymerase chain reaction(rt-qPCR) to detect the expression of RSPO-1and Lgr5mRNA. To compare the mast cells and its RSPO-1expression level with clinic pathological features.Results:The expression rates of RSPO-1in colorectal carcinoma, para-carcinoma and proximal surgical margin tissues is90.48%,100%and80.95%, the difference was statistically significant (P<0.0001) with Immunohistochemical; the expression rates of C-kit in colorectal carcinoma, para-carcinoma and proximal surgical margin tissues were28.57%,100%and80.95%, the difference was statistically significant (P<0.0001); the strongly positive (++) expressing of C-kit in para-carcinoma tiusses of tumor differentiation and lymph node metastasis was positively correlated sub-station case (P=0.02and0.015), while the strongly positive (++) expression of RSPO-1in carcinoma or paraa-carcinoma tiusses of tumor differentiation, lymph node metastasis, depth of invasion and staging no significant correlation (P values were0.194,0.114,0.5and0.44); The expression of Lgr5in colorectal carcinoa is higher than para-carcinoma and proximal surgical margin tissues with real-time quantitative polymerase chain reaction and Western blot. Conclusion:The Lgr5was significantly over-expression in carcinoma tissues, the infiltrating mast cells express RSPO-1in human colorectal carcinoma environment; the density of infiltrating mast cells and RSPO-1in colorectal carcinoma enviroment were associated with tumor differentiation and lymph node metastasis. Therefore, the RSPO-1expressed by infiltrated mast cells in colorectal carcinoma blinding to colorectal carcinomastem cell surface receptor Lgr5may be associated with maintaining the stability and malignant biological effects of of colorectal carcinoma stem cell. Part Ⅱ The mast cells involved in maintaining the stemness of mouse colonrectal cancer stem cellsObjectives:To investigate the mechanisim of mast cell mediated RSPO-1affectting Wnt/β-catenin signaling pathway in mouse Lgr5+colorectal stem cell:CT-26(wt).Methods:The cultured mouse infiltrating mast cells P815, mouse colon carcinoma cells CT-26; CT-26cells were cultured in serum-free medium from spheroid cells:RNA was extracted stem cells detection. The P815cells were stimulated with different concentrations of SCF suspension culture, cell proteins were extracted RSPO-1expression with Western blot. The CT-26spheroid cells and P815cells serum-free suspension co-culture of different interventions, to observe the cells’s growth and the total mRNAs and protein of CT-26spheroid cells were extracted; The expression of β-catenin, TCF1/4, c-Myc mRNA and protein from the CT-26spheroid cells with the Real-time PCR and Western-blot.Results:Western-Blot show infiltrating mast cell line P815after receiving lOng/ml of SCF stimulation after48hours, the expression of RSPO-1was significantly increased, but with an increase in the concentration of SCF and increased stimulation time, the expression of RSPO-1didn’t have a significantly increasing trend; Gremlin and Noggin didn’t expressed after the SCF stimulation; the CT-26cells with RSPO-1stimulation,the expression of stem cell surface markers CD133+and SOX2was significantly higher than unstimulated or blocking RSPO-1cells with Real-time PCR; Western-Blot showed:CT-26spheroid cells and P815cells were suspended after co-culture,the Wnt/β-catenin signal pathway was activitated, the β-catenin, Tcf4and c-Myc protein with in the cytoplasm and the nucleus were significantly higher than the unstimulated cell and blocking group.Conclusion:Mast cells mediated RSPO-1can stimulate colonrectal cancer stem cells, its Wnt/β-catenin signal pathway was activation, β-catenin which was accumulated in the cytoplasm went into the nucleus activating the transcription factor TCF/Lef family. Therefore, mast cells mediated RSPO-1played an important role in maintaining the sternness of mouse colonrectal cancer stem cells. Part Ⅲ The mast cells affecting Wnt/β-catenin signaling pathway in mouse Lgr5+colitis and colorectal cancer model.Objectives:To investigate the mechanisim of mast cell mediated RSPO-1affecting Wnt/β-catenin signaling pathway in mouse Lgr5+colitis and colorectal cancer model.Methods:Balb/c female mice with colon cancer induced by classical chemical method (AOM+2%DSS). After observing the growth of mouse, we detected the expression of β-catenin, TCF4, c-Myc gene in the8weeks,16weeks and32weeks with immunohistochemistry and flow cytometry Lgr5+mouse colon cancer tissues. The expression of RSPO-1in mouse colon cancer, colitis and normal tissues with Western-blot.Results:20%(2/10) mouses were successfully established model of colorectal cancer in the32weeks; The mortality rates of the Balb/c mice which were injected with AOM and drinking2%DSS were20%(1/10) and40%(4/10) in2and4weeks; In ontrast with normal or inflammation(DSS induced only) group, percentage of Lgr5+colonic epithelial cells (CD45-CD31-CD326+) and Wnt/β-catenin signaling pathway activation levels; the expression of p-catenin, TCF, c-Myc in AOM+2%DSS group was significantly higher than normal and DSS groups, the infiltration mast cells located in the edge of the growth of colon carcinoma invasion, the expression of RSPO-1protein was significantly increased.Conclusions:In the mouse model of colon cancer,mast cells mediated RSPO-1continuallystimulated the activation of Wnt/β-catenin signaling pathway in the Lgr5+colonrectal cancer.