Correlation Between HMGB1 And Cervical Cancer Metastasis

ObjectiveHigh mobility group box 1 (HMGB1) has been confirmed to be closely related with tumorigenesis, metastasis and angiogenesis in a variety of malignancies such as pancreatic cancer, colon cancer, breast cancer and prostate cancer, etc. Recent findings have supported the hypothesis that HMGB1 is correlated with lymph vessel density in the primary tumors and VEGF-C expression is positively correlated with HMGB1 expression. Cervical cancer is a kind of tumor with strong metastatic, and lymph node metastasis occurs in the early stage. In the previous studies, we have confirmed that overexpressed HMGB1 is involved in the invasion and lymph node metastasis in cervical squamous cell carcinoma (CSCC); HMGB1mRNA and protein expression was inhibited, HeLa cell growth slowed and the cell cycle was arrested in the G2 phase after HMGB1siRNA plasmid transiently was transfected into HeLa cells. This study was to evaluate effects of HMGB1 expression suppressed by siRNA on cell proliferation, invasion and migration of human cervical cancer cell line HeLa, and to preliminary explore the role of exogenous HMGB1 in lymphangiogenesis to provide a theoretical basis for the possible role of HMGB1 in promoting tumor lymphangiogenesis and the subsequent lymphatic metastasis.Methods1. HMGB1siRNA eukaryotic expression vector PGCsi3.0-1 and the negtive control vector PGCsi3.0-Neg have been previously constructed. The vectors were transfected into HeLa cells by lipofectamine2000, and then the successfully transfected single cell was selected by G418 and amplified.2. HMGB1 expression was detected by RT-PCR and Western blot.3. MTT was used for observing HeLa cell proliferation in vitro.4. HeLa cell invasion in vitro was evaluated by a transwell chamber model.5. HeLa cell migration in vitro was evaluated by cell scratch method.6. Human lymphatic endothelial cells (LECs) were treated with different concentrations of human rHMGB1 or rVEGF-C. Changes in cell proliferation in vitro was assessed by MTT assay.7. LEC migration in vitro was assessed by a transwell chamber assay.8. LEC capillary-like tube fonnation in vitro was assessed by a Matrigel model.Results1. The vectors were transfected into HeLa cells successfully, and the highest transfection efficiency occurred at 48h after transfection. HeLa cell lines with HMGB1 knocked down were screened by G418.2. The introduction of PGCsi3.0-1 efficiently and specifically inhibited the expression of HMGB1 mRNA and protein.3. HeLa cell growth slowed down, cell migration rate was inhibated the and the number of penetrating cells decreased by RNA interference.4. Human rHMGB1 induced LEC proliferation, migration and tube formation in a dose-and time-dependent manner. As a specific lymphangiogenes factor, the role of rVEGF-C in promoting lymphangiogenesis was significant.Conclusion1. HMGB1 expression could be inhibited successfully by RNAi induced by eukaryotic expression vector PGCsi3.0-1/HMGB1 siRNA. This consequently inhibited the proliferation, migration and invasion in vitro of human cervical cancer cell line HeLa effectively. This study provided a preliminary result in searching of RNAi therapy for cervical cancer.2. HMGB1 and VEGF-C are lymphangiogenic factors which can accelerate lymphatic metastasis of tumor cells by promoting lymphangiogenesis. This provides new theoretical basis for the metastasis mechnisms of cervical cancer.