The Study Of HMGB1's Effect On Lymphangiogenesis And The Mechanism In Colon Cacer

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.HMGB1 is found to be important in the transcription regulations as a small molecular DNA binding protein firstly. In recent years, it was found having many extra cellular functions. After binding with its receptor RAGE, it plays an important role in inflammation, cell migration, differentiation, tumorigenesis etc. In vivo, HMGB1 derives mainly from the damaged or necrosised cells, and macrophages actived by hypoxia, endotoxin, TNF-α,IL-1β. Meanwhile HMGB1 also can feed back to induce macrophages to secrete many factors such as VEGF, TNF-α,IL-8.Colon cancer, a common malignant tumor in digestive system, its metastasis is mainly through lymphatic. Several researches have shown that HMGB1 could affect the angiogenesis in tumor, but its role in lymphangiogenesis is still unknown. In this study we detected HMGB1's effect on VEGF-C/D in order to unveil its contribution in colon cancer lymphangiogenesis and lymphonode metastasis and its potetial mechaninsm, which may laid a foundation for further understanding the mechaninsm 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 VEGF-C/D, HMGB1, macrophage and lymphantic with immunohistochemical analysis. The results were analyzed statistically to investigate the correlations of VEGF-C/D, HMGB1, lymphonode metastasis, lymphatic counts, TAMs counts and some other clinical pathology characterictics. 2. HMGB1's effect assayAfter stimulated by HMGB1, the changes in mRNA and protein of VEGF-C/D was detected in conlon cancer cell line HCT116 and mononuclear macrophage line U937 by RT-PCR and western blot. RNAi vector, carrying siRNA targeting NF-κB, 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 HMGB1 affect VEGF-C/D.3. HMGB1's effect on tumor lymphangiogenesisThe recombinant expression vector of HMGB1-pLxsn was constructed and transfected into HCT116 cell line, then transplanted into nude mouses. After harvested the transplanted neoplasm, the expression of VEGF-C/D, the lymphantic and lymphonode metastasis in the transplantation tumor were dectected and compared with the control group.Results1. Colon cancer tissue immunohistochemistry assayVEGF-C/D were detected in both cancer cells and tumor associated macrophages. HMGB1 and VEGF-C 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/D was consistent with the expression of HMGB1. HMGB1, VEGF-C are closely correlated with lymphatic counts and TAMs counts, but not with patient's age, sexual, cellular differentiation.2. HMGB1's effect assayBased on the RT-PCR and western blot results, data demonstrated that the expression of VEGF-C had a dose-dependent relationship wiht HMGB1. Results also showed that stimulated with HMGB1, the expression of VEGF-D reached its peak at 4 hours and the time for VEGF-C was 12 hours. Interesting, after transfected the siRNA vector targeting NF-κB, the stimulation of HMGB1 on VEGF-C/D was suppressed.3. HMGB1's effect on tumor lymphangiogenesisIn the transplanted tumor, the lymphatic counts in transfected group was significantly higher than that in the control group (P<0.05).ConclusionAfter studying the HMGB1's effect on VEGF-C/D expression and its mechanism in colon cacer, we hypothesized (1) VEGF-C/D were expressed not only in conlon cacer cell but also in TAMs, and they could affect each other. (2) HMGB1 can stimulate the expression of VEGF-C/D in colon cancer and macrophage. (3) HMBGB1 can affect VEGF-C/D through the NF-κB signaling pathway. (4) HMGB1 can induce lymphangiogenesis in colon caner and further affect the lymphatic metastasis by its effct on VEGF-C/D in local microenvironment.