The Study Of Tiam1 Gene's Silencing On Lymphangiogenesis In Colon Cancer

Background and ObjectiveInvasion and metastasis are the basic characteristic of malignant tumor and the main lethal cause. Lymphatic is the most common metastasis way of the majority cancers. Having lymphonode metastasis or not is an important index of prognostic. With the finding of the specific markers of lymphatic and lymphangiogenesis factors, tumor cells had been reported that they could induce lymphangiogenesis by autocrine or paracrine VEGF-C/D.Tiam1 was separated from the T lymphoma of mice in 1994. It was only in testicle and brain. Recently more and more studies reported that it was related to migration and metastasis in breast cancer, rhinopharynx cancer and other cancers. The mechanism is regarded that Tiam1 promote tumor cells migration and adherence through activation of Rac1.Colon cancer, a common malignant tumor in digestive system, its metastasis is mainly through lymphatic. Several researches have shown that Rac1, the backward position of Tiam1, could affect the angiogenesis in tumor, but Tiam1's role in lymphangiogenesis is still unknown. In this study we detected Tiam1's effect on VEGF-C/D in order to unveil its contribution in colon cancer lymphangiogenesis and lymphonode metastasis and its potential mechanism, which may laid a foundation for further understanding the mechanism of lymphatic metastasis route in colon cancer.Methods1. Colon cancer tissue immunohistochemistry assayHuman colon cancer samples, obtained from southwest hospital surgery department, were sliced and detected Tiam1, Rac1, VEGF-C/D and lymphantic with immunohistochemical analysis. The results were analyzed statistically to investigate the correlations of Tiam1, VEGF-C/D, lymphonode metastasis, lymphatic vessel density and some other clinical pathology characteristics. 2. Tiam1's effect assayAfter Tiam1 silencing, the changes in mRNA and protein of VEGF-C/D was detected in conlon cancer cell line HCT116 by RT-PCR and Western blot. RNAi vector, carrying siRNA targeting Tiam1, was constructed, and transfected into HCT116 cell line. And the expression of VEGF-C/D in transfected cells and control group cells was compared to get some information about the mechanism that Tiam1 affects VEGF-C/D.3. Tiam's effect on tumor lymphangiogenesisThe plasmid of pGenesil-1.neo.EGFP/Tiam1-RNAi was constructed and transfected into HCT116 cell line, then transplanted into nude mouses. After harvested the transplanted neoplasm, the expression of Rac1, VEGF-C/D, LMVD and lymphonode metastasis in the transplantation tumor were dectected and compared with the control group.Results1. Colon cancer tissue immunohistochemistry assayTiam1/Rac1 and VEGF-C/D were detected in cancer cells . Tiam1/Rac1 and VEGF- C/D were much higher in lymphonode metastasis group than that in non-metastasis group (P<0.05). Furthermore, the data statistics suggested that the expression of VEGF-C was consistent with the expression of Tiam1. Tiam1 and VEGF-C are closely correlated with lymphatic vessel density, but not with patient's age, sexual, cellular differentiation.2. Tiam1's effect assayBased on the RT-PCR and Western blot results, data demonstrated that the expression of VEGF-C/D had a positive relationship wiht Tiam1. After transfected the siRNA vector targeting Tiam1, the expression of VEGF-C/D was suppressed.3. Tiam's effect on tumor lymphangiogenesisIn the transplanted tumor, the lymphatic microvessel density in transfected group was significantly lower than that in the control group (P<0.05).ConclusionAfter studying the Tiam1's effect on VEGF-C/D expression and its mechanism in colon cancer, we hypothesized (1) Tiam1/Rac1 and VEGF-C/D were expressed in conlon cancer tissue, and Tiam1 was significantly correlated with Rac1, VEGF-C, LMVD and metastasis. (2) The plasmid of Tiam1-RNAi was constructed successfully, and verified by enzyme incision and sequencing. (3) Based on the RT-PCR and Western blot results, the expression of Tiam1 was suppressed after transfected the plasmid of Tiam1-RNAi. (4) In vitro Tiam1 gene's Silencing can reduce the expression of VEGF-C/D in colon cancer cells. Tiam1 maybe affect VEGF-C/D through the Rac1 signaling pathway. (5) Tiam1 can induce lymphangiogenesis in colon cancer and further affect the lymphatic metastasis by its effect on VEGF-C/D in local microenvironment.