IL-6通过miRNA调控Twist诱导胆囊癌CD133+CD44+肿瘤干细胞增殖的机制研究

Is chronic cholecystitis the high risk factor of gallbladder cancers? Our preliminary experiment suggested that IL-6 which was highly expressed in the stroma of gallbladder cancers not only elevated the protein expression of Twist Gene, but also induced the proliferation of CD133+CD44+ Cancer stem cells of gallbladder cancers. By performing micro RNA microarray analysis we found that IL-6 down-regulated the expression of miR-33a and Twist gene was the target of miR-33a. This study plan to verify that ①miR-33a mediates the post-transcriptive regulation of IL-6 towards Twist;②Twist mediates the pro-proliferation effets of IL-6 towards gallbladder cancer stem cells by performing RNA interference, slow virus transfection, contrusting Twist gene 3'-UTR and fluorescent report gene complex and so on. This research explores the mechanisms between IL-6 and cancer stem cells of gallbladder cancers in the level of micro RNA and signal transduction which may provide experimental evidence to identify the correlation between chronic cholecystitis and gallbladder cancers.

慢性胆囊炎是胆囊癌发生的高危因素吗？本研究前期发现胆囊癌较慢性胆囊炎间质中IL-6表达明显增高。实验还发现IL-6可提高Twist蛋白表达，同时诱导CD133+CD44+胆囊癌肿瘤干细胞增殖，从而提高胆囊癌细胞种植裸鼠成瘤能力。通过micro RNA芯片筛选和分析发现IL-6下调了miR-33a的表达，Twist是miR-33a作用的靶基因。本研究拟通过RNA干扰、慢病毒转染、构建3ˊ-UTR与下游荧光素报告基因复合体等方法证明①miR-33a介导了IL-6对Twist基因的转录后水平调控；②Twist介导了IL-6对胆囊癌肿瘤干细胞的增殖作用。本研究将从miRNA水平和信号转导角度阐明IL-6通过miR-33a调控Twist诱导胆囊癌干细胞增殖的机制，为明确慢性胆囊炎与胆囊癌的相关性提供实验依据。

本课题通过对临床胆囊癌标本检测发现，IL-6与EMT相关基因mRNA表达水平相关。较癌旁组织，癌组织中IL-6，Twist及Vimentin蛋白及mRNA表达水平较高，而E-cadherin表达较低，且IL-6，Twist及E-cadherin的表达与分化程度，局部浸润，淋巴结转移及TNM分期密切相关。进一步生存分析，IL-6和Twist高表达组胆囊癌患者生存时间较低表达组明显缩短（P＝0.0468， P＝0.0103），而E-cadherin高表达组生存时间较低表达明显增加（P＝0.0145）。利用CCK8细胞增殖实验，Brdu增殖实验，免疫荧光及流式实验，克隆形成实验，transwell迁移侵袭实验，划痕实验，动物皮下成瘤模型及腹腔转移模型等实验证明IL-6能够促进胆囊癌细胞增殖、侵袭及发生上皮间质转化，上调转录因子Twist1及Vimentin蛋白表达，下调E-cadherin表达下降。免疫沉淀共沉淀发现IL-6与Twist结合。（P<0.05）。通过芯片筛查IL-6刺激胆囊癌细胞及QRT-PCR验证发现miR-33a表达下调明显，并且miR-33a与Twist表达相关，上调miR-33a抑制细胞增殖、迁移及侵袭能力，EMT相关转录因子Twist和标记物Vimentin表达下降，而E-cadherin 表达升高，同时细胞内源性IL-6分泌量下降。机制研究发现miR-33a靶向调节Twist表达。IL-6作用细胞抑制miR33a表达，下调的miR33a可促进twist的上调。体内实验证实稳定过表达miR-33a可抑制皮下及腹腔成瘤能力。此外，我们也在临床标本中证实miR-33a表达较癌旁组及慢性胆囊炎组低。miR-33a的表达与IL-6，Twist及Vimentin均呈负相关，与E-cadherin表达呈正相关。miR-33a的表达与胆囊癌局部浸润（P=0.005），淋巴结转移（P=0.001）及TNM分期（P=0.000）密切相关。患者生存期与miR-33a表达（P＝0.021），胆囊癌局部浸润（P＝0.021），淋巴结转移（P＝0.073），TNM分期有关（P＝0.026）。通过以上结果表明IL-6 通过 miR-33a 调控 Twist 诱导胆囊癌肿瘤增殖及侵袭。