Experimental Study Of The Changes Of Wnt Signaling Pathway Effecting Tumor Angiogenesis In Colon Cancer Cells With CD151 Deletion

Colorectal cancer(colorectal cancer, CRC) is one of the most common malignant tumors at present, and the age of onset is becoming younger and younger, which may be related to the gradual Western diet and lifestyle. In recent years, with the improvement of diagnostic techniques and treatment, the survival of patients with colorectal cancer is significantly prolonged, but the advanced colorectal cancer is still one of the main causes of cancer related deaths in China. So how to inhibit the development of tumor has become an urgent problem to be solved. People found that angiogenesis play an important role in the process of tumor recurrence and metastasis, in which CD151- integrin complex can promote extracellular signal inward, activate intracellular pathways and improve blood vessel hyperplasia. In recent years, Wnt signaling pathway closely related to tumor has attracted extensive attention, and it can affect tumor angiogenesis by regulating VEGFs, but whether Wnt signaling pathway can mediate CD151 regulateing tumor angiogenesis, it is not clear at present.Objective: In this experiment, human colon cancer cells(HT29) were used as a reference control, and the gene and protein expression changes of the ?-catenin were tested in CD151 gene knockout HT29 cells(CD151--HT29), and through comparatively observating growth and vascular proliferation differences of two kinds cells tumour in nude mice, we explored the mechanism that the expression change of CD151 gene affect the angiogenesis through Wnt/?-catenin signaling pathway in colon cancer.Methods:1 Using of cell culture techniques, HT29 cells and CD151- HT29 cells were adherent to culture to observe the growth stated and to select the logarithmic growth phase HT29 cells and CD151- HT29 cells for cell experiments and animal modeling after CK20 detection.2 Western blotting were used to detecte the expression of CD151 protein in HT29 cells and CD151- HT29 cells.3 Real-Time PCR were used to detect the expressing of ?-catenin m RNA in HT29 cells and CD151- HT29 cells, and we explored CD151 gene deletion state how to impact ?-catenin gene transcription level.4 Western blotting were used to detecte the expression of ?-catenin protein in HT29 cells and CD151- HT29 cells, we explored whether CD151 protein levels can affect the function of the Wnt signaling pathway.5 The experimental mice were randomly divided into two groups, which were defined as HT29 cells group and CD151- HT29 cells group, the cells were inoculated subcutaneously in nude right armpit to establish xenograft animal model. We observed daily the survival status of nude mice and tumor formation time and so on, measured tumor volume every three days and drew growth curve. The nude mice were killed in 21 days, and we compared the differences in their tumor formation and tumor angiogenesis.Results:1 Western blotting techniques were used for the experiment HT29 cells and CD151- HT29 cells, and the results showed that CD151 protein was expressed in HT29 cells, but absent in CD151--HT29 cells.2 Real-Time PCR techniques were used to detect HT29 cells and CD151--HT29 cells, the results showed that ?-catenin m RNA CT:(14.65 + 1.23) and(14.05 + 1.16) in the two kinds of cells. Using HT29 cells as control group, CD151--HT29 as the experimental group,we calculated 2-??CT <1, ?-catenin transcription level of m RNA in HT29 cell was significantly higher than that of CD151--HT29 cells. Results had significant difference(P < 0.05).3 Western blotting techniques were used for the experiment HT29 cells and CD151-- HT29 cells, and the results show the expression of ?-catenin protein in HT29 cells was higher than CD151--HT29 cells, the fluorescence intensity value of its protein expression were(0.186 ± 0.009) and(0.168 ± 0.010). Results had significant difference(P < 0.05).4 tumor detection(1) HT29 cells group and CD151--HT29 cells group tumor weights were(1.175 ± 0.127) and(1.059 ± 0.142), the results were statistically significant difference(P <0.05), described CD151--HT29 cells tumor growth was inhibited.(2) HT29 cell group and CD151--HT29 cell transplantation tumor tissue were staind by CD34,we calculated the MVD value as follows: HT29 cells group(42.20±5.36), CD151-HT29 cells group(32.80±5.89),the results were statistically significant difference(P <0.05).Conclusion:1 HT29 tumor cell proliferation was significantly higher than the CD151 gene knockout HT29 cells, suggesting that down-regulated CD151 gene expression contributed to inhibit the growth of colon cancer cells.2 Knocking CD151 gene HT29 cells in tumor tissues significantly reduced tumor angiogenesis, it suggested that expression of CD151 gene maybe involve the regulation of tumor angiogenesis.3 The downregulation of CD151 gene expression accompanied with the decrease of the level of intracellular ?-catenin protein, it suggested that Knocking CD151 gene may inhibit tumor angiogenesis and tumor cell proliferation by regulating the function of Wnt signaling pathway.