| Background:Bladder cancer (BC) was the most common genitourinary malignancy of the world, it was important to predict the invasive behavior of BC in clinical work, and specific molecular markers which could serve as credible progressive and prognostic factors were needed. High mobility group box (HMGB) proteins, include HMGB1and HMGB2were ubiquitous, abundant nuclear proteins and had diverse functions in the cell, such as binding to DNA without sequence specificity and regulating transcription, replication, DNA repair and recombination. Overexpression of HMGB1and HMGB2had been observed in several human cancers, and exhibited important effects in mediation of tumor growth, angiogenesis, metastasis and chemotherapy resistance. But less was known of the relevance of HMGB1and HMGB2in BC carcinogenesis, such as their angiogenic effects and precise signaling pathways.Objective:To understand the role of HMGB in BC, we investigated the expressions and effects of HMGB1and HMGB2in BC tissues, then studied the effects of lentivirus-mediated suppression of HMGB1expression on BC cells and explored possible downstream mechanisms in vitro and in vivo.Methods:(1) In this study,15cases of normal bladder tissues and64cases of BC tissues were collected, immunohistochemical analysis and Real-time PCR were used to demonstrate the expression of HMGB1and HMGB2in human BC. The relationships between HMGB1and HMGB2expressions and clinicopathologic parameters were analyzed. The relationships between HMGB1and HMGB2expressions and vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) were also analyzed.(2) The lentivirus vectors containing a small hairpin RNA (shRNA) to target HMGB1were constructed. After5637human BC cells were infected, Real-time PCR and Western-blot were used to measure HMGB1expression. The influence of HMGB1on the proliferation of BC cells was assessed by MTT, BrdU and colony formation. Migratory and invasive abilities were determined by using of the Transwell assay. Cell cycle was analyzed with flow cytometric assay.(3) To assess the influence of the tumorigenicity by inhibiting HMGB1gene expression in BC cells, we injected shRNA-HMGB1lentivirus into the tumors of the nude mice transplanted models and measured the tumors’sizes and weights. Western-blot was performed to detect NF-KB/p65and VEGF-C expression after transfection of sh-HMGB1into5637BC cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of VEGF-C influenced by the changes of NF-κB/p65activities.Results:(1) Overexpressions of HMGB1were determined in55%of64cases and30%for HMGB2at the same time by immunohistochemical analysis. Overexpressions of HMGB1and HMGB2were significantly associated with clinical stage and pathology grade of tumors (p<0.05). HMGB1and HMGB2were also associated with VEGF expression and MVD (p<0.05).(2) HMGB1mRNA relative level (0.25±0.04) and protein expressing in the sh-HMGB1group were significantly inhibited by RNAi (p<0.05). The sh-HMGB1group displayed an increased proportion of cells (63.44±1.17%) in the G0/G1phase compared with the Con group (53.91±1.09%) and sh-Con group(51.13±1.33%)(p<0.05). Compared with the Con and sh-Con groups, the sh-HMGB1group displayed a significant cell proliferation defect by MTT and BrdU assay and the number of colonies (51.00±13.11) was significant decreased (p<0.05). The Transwell assay revealed that the sh-HMGB1group cells had much lower migratory ability and invasive activity (p<0.05).(3) On the day30, the tumor volume (285.90±86.15mm3) and weight(0.26±0.11g) of the LV-HMGB1group were less than the NS group and LV-Con group after the shRNA-HMGBl lentivirus was injected into the tumuors of the nude mice transplanted models (p<0.05). Western-blot analysis showed that the protein levels of NF-κB/p65and VEGF-C of sh-HMGB1group were significantly decreased (p<0.05). ELISA showed that the VEGF-C concentration was significantly decreased in the sh-HMGB1group (211.58±8.16pg/ml)(p<0.05). The NF-κB inhibitor PDTC could decrease the expression of VEGF-C, and HMGB1as a NF-κB agonist could increase the expression of VEGF-C significantly (p<0.05) at the same time.Conclusion:(1) Overexpressions of HMGB1and HMGB2in the BC tissues indicated that they exhibited important effects in mediation of tumor growth, angiogenesis, metastasis and etc.(2) Specific lentivirus-mediated knockdown of HMGB1significantly deceased the HMGB1expression in the BC cells, impacted the cell cycle and deceased the proliferative, migratory and invasive abilities of the BC cells.(3) Injecting sh-HMGB1lentivirus could decrease the BC cells proliferation and made the BC cells nearly lost their tumorigenicity in nude mice models. Downregulation of HMGB1could inhibit the expression of NF-KB/p65and VEGF-C in the BC cells. Meanwhile, NF-κB activity could modulate the concentration of VEGF-C. HMGB1regulated VEGF-C expression via the NF-κB signaling pathway possibly.These results provided new evidence of an important role for HMGB1in the development of BC, suggested that lentiviruses delivering shRNA against HMGB1might be a promising tool for bladder cancer therapy. |
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