siRNA干扰HMGB1表达对膀胱癌T24株细胞恶性生物学行为的影响及其可能的机制

Effects of siRNA interfering expression of HMGB1 on malignant biological behaviors of bladder cancer T24 line cell and its possible mechanism

目的:探讨高迁移率族蛋白1(high-mobility group box 1,HMGB1)对膀胱癌T24株细胞的增殖、凋亡及恶性生物学行为的影响及其潜在作用机制。方法:收集2014年12月至2016年1月期间重庆医科大学附属第一医院泌尿外科病区手术切除的20例膀胱癌以及相应癌旁组织,免疫组化方法检测膀胱癌和癌旁组织HMGB1表达差异;应用RNAi处理T24细胞并分为空白对照组(Blank)、阴性对照组(NC)和干扰组(si HMGB1);CCK-8、流式细胞术、划痕实验和Transwell侵袭实验分别检测HMGB1敲低后对T24细胞增殖、凋亡、周期、迁移以及侵袭能力的影响;Western blotting检测不同膀胱癌细胞株BIU-87和T24细胞的HMGB1表达水平以及敲低HMGB1对T24细胞恶性生物学行为相关蛋白的影响。结果:与癌旁组织相比,HMGB1在膀胱癌组织处于高表达状态[(67.33±4.91)vs(12.00±3.79),P<0.05]。与NC组和Blank组相比,si HMGB1组细胞增殖受抑制(P<0.05);流式细胞术提示敲低HMGB1后细胞凋亡率增加,细胞周期阻滞在G0/G1期;划痕实验及Transwell侵袭实验显示,敲低HMGB1后细胞迁移(P<0.05)以及侵袭[穿膜细胞数:(16.33±1.45)vs(35.00±1.53)、(34.00±2.08)个,均P<0.05]能力减弱;Western blotting结果显示敲低后E-钙黏着蛋白表达上调(P<0.05),N-钙黏着蛋白、波形蛋白、基质金属蛋白酶(metrix metalloproteinase,MMP)-2、MMP-9、细胞周期蛋白D1、c-Myc、β-联蛋白表达下调(均P<0.05)。结论:HMGB1可通过促进膀胱癌细胞EMT进而增强其恶性生物学行为,其机制可能是通过β-联蛋白信号通路介导。

Objective：To explore effects of high-mobility group box 1 （HMGB1） on proliferation,apoptosis and malignant biological behaviors of bladder cancer T24 cell line as well as its potential mechanism.Methods：Twenty cases of surgically resected bladder cancer and corresponding adjacent normal tissues form the patients who were hospitalized in Department of Urinary Surgery,the 1 st Hospital affiliated to Chongqing Medical University during December 2014 to January 2016 were collected.The T24 cells were treated by RNAi technique and divided into blank control group,negative control （NC） group and interfere （siHMGB1） group.Differences of HMGB1 expression between bladder cancer and adjacent normal tissues were detected by immuno chemistry assay.CCK-8 assay,flow cytometry assay,scratch test and Transwell invasion test were used to examine effect of knocking down HMGB1 on abilities of proliferation,apoptosis,cell cycle,migration and invasion of the T24 cells respectively.Western blotting assay was used to detect expression levels of HMGB1 in BIU-87 and T24 cells and effect of knocking down HMGB1 on expression of malignant biological behavior related proteins in the T24 cells.Results：Expression of HMGB1 in the bladder cancer tissues was obviously higher than that in the adjacent normal tissues （[67.33 ± 4.91] vs [12.00 ± 3.79],P 〈 0.05）.Comparing with the negative control and blank control groups,proliferation of the T24 cells was inhibited in the siHMGB1 group （P 〈0.05）.Results of flow cytometry assay suggested that apoptosis rate of the T24 cells increased and its cell cycle arrested in phase of G0/G1 after knocking down HMGB1;results of scratch and Transwell invasion tests showed that after knock-down of HMGB1,abilities of migration （P 〈0.05） and invasion （[16.33 ±1.45] vs [35.00 ±1.53],[34.00 ±2.08],P〈0.05] of the T24 cells decreased.Results of Western blotting assay showed that after the knock-down,expression of E-cardherin protein was upregulated,expressions of N-cadherin,vimentin,metrix metalloproteinase（MMP）-2,MMP-9,cyclinD1,c-Myc and β-catenin proteins were down-regulated （all P 〈 0.05）.Conclusion：HMGB1 could promote epithelial-mesenchymal transition （EMT） of the bladder cancer cells and then enhance their malignant biological behaviors.The mechanism might be mediated by β-catenin signaling pathway.