| Background:Bladder cancer is one of the most common malignant tumors of genitourinary system, about90%of which are bladder urothelial carcinoma (BUC). Although BUC has been extensively studied for many years, the etiology and molecular biological mechanism of BUC are still not entirely clear. Meanwhile, the efficacy of therapeutic methods remains unsatisfactorily and the overall prognosis of BUC patients is still far from optimistic. Therefore, efforts to clarify the molecular biological mechanisms of BUC occurrence and progression are quite meaningful to improve diagnosis and prognosis of this deadly disease.High-mobility group box1(HMGB1) is a highly conservative nuclear non-histone protein, which has been generally found in eukaryotes. Previous studies have demonstrated that HMGB1involves in a variety of cancer processes, such as unlimited cell proliferation, apoptosis, angiogenesis, metastasis and invasion, which plays an important role in the development of human malignancies. However, until now there has been little information about the expression and function of HMGB1in BUC.Objectives:(1) To investigate the expression of HMGB1in human BUC tissue and cell with regard to related clinicopathologic significance. (2) To explore the construction and identification of plasmid vector with HMGB1short hairpin RNA (shRNA) and transfected human BUC cell line T24with lipofectamineTM2000to specifically down-regulate the expression of HMGB1.(3) To analyze the effects of RNA interference (RNAi) on the expression of HMGB1and the bioactivity of T24cell line.(4) To elucidate the potential downstream mechanism of HMGB1to BUC cell.Methods:(1) Thirty cases of BUC tissue and matched normal adjacent tissue were collected. Western-blot and real-time polymerase chain reaction (Real-time PCR) were performed to detected the expression of HMGB1. Meanwhile, the location of HMGB1protein was detected by immunohistochemisty. The relationships between clinicopathologic parameters and HMGB1expression were also studied.(2) The pGPU6/GFP/Neo-plasmid vector with HMGB1shRNA was constructed and transfected T24cell with lipofectamineTM2000. Western-blot and Real-time PCR were used to measure the impacts of RNAi on HMGB1expression.(3) The effects of HMGB1knockdown on T24cell proliferation, apoptosis, migration, invasion and cell cycle were assessed by MTT assay, transwell assay and flow cytometry, respectively.(4) Western-blot and Real-time PCR were performed to measure nuclear factor kappa B/p65(NF-κB/p65) and vascular endothelial growth factor-C (VEGF-C) expression after down-regulation of HMGB1. The location of NF-KB/p65protein was detected by indirect immunofluorescence assay, and the activity of NF-κB/p65protein was measured by enzyme-linked immunosorbent assay (ELISA).Results:(1) Both protein and mRNA expression levels of HMGB1were significantly up-regulated in BUC compared with matched normal adjacent tissue. HMGB1protein immunoreactivity was mainly locateded in the nuclei of BUC, and was rarely presented in the cytoplasm; furthermore, the HMGB1expression levels had a significantly positive correlation with the clinicopathologic parameters; meanwhile, cellular HMGB1expression increased from5637, BIU-87to T24cell line, in accordance with their malignant potential.(2) We successfully constructed three plasmid vectors with shRNA targeting HMGB1and transfected vectors into T24cell with lipofectamineTM2000. All the specific shRNA sequences could significantly inhibit the expression of HMGB1, with inhibition by HMGB1shRNA-539being highest.(3) Knockdown of HMGB1significantly suppressed T24cell proliferation, migration, invasion, and induced cell cycle arrest at the G0/G1phase as well as increased cell apoptosis.(4) Knockdown of HMGB1decreased the expression of NF-KB/p65and VEGF-C, which was accompanied with the suppression of NF-κB/p65nuclear translocation and activity.Conclusions:(1) HMGB1protein and mRNA have a significantly high expression in BUC tissue and cell, and its overexpression positively relates with tumor grade and stage. HMGB1may associate with development and progression of BUC.(2) Knockdown of HMGB1can significantly suppress BUC cell proliferation, migration, invasion, and induce cell cycle arrest at the G0/G1phase as well as increase cell apoptosis. HMGB1may serve as a potential therapeutic target.(3) Knockdown of HMGB1can obviously decrease the expression of NF-κB/p65and VEGF-C, which is accompanied with the suppression of NF-κB/p65nuclear translocation and activity. HMGB1may regulate VEGF-C expression via the NF-κB/p65signaling pathway. Figures:25; tables:35;references:135. |
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