乙型肝炎相关性肝癌中Toll样受体4调控周期蛋白依赖激酶4/6对其增殖的影响  

Toll-like receptor 4 regulates hepatitis B virus related hepatic carcinoma cell proliferation by controlling cyclin-dependent kinase 4/6 expression

作　　者：周勇[1,2] 黄文峰[2] 冯潜[1] 石世代 李恩亮[1] 李科浩 吴荣寿[1] 邬林泉[1] 

机构地区：[1]南昌大学第二附属医院肝胆外科,江西省南昌市330006 [2]萍乡市人民医院普外科,江西省萍乡市337055

出　　处：《世界华人消化杂志》2015年第19期3029-3037,共9页World Chinese Journal of Digestology

摘　　要：目的:探讨Toll样受体4(Toll-like receptor 4,TLR4)对乙型肝炎相关性肝癌细胞增殖的影响及作用机制.方法:Western blot法检测36例乙型肝炎相关性肝癌患者组织及相应癌旁组织中TLR4、周期蛋白依赖激酶(cyclindependent kinase,CDK)4和CDK6的蛋白表达水平,并分析癌组织中TLR4与CDK4,CDK6之间的关联性.构建3个特定干扰TLR4的siRNA序列和一个阴性对照序列,通过脂质体转染肝癌细胞Hep-3B,Western blot法筛选出最佳干扰序列.Western blot法检测转染前后肝癌细胞中TLR4、CDK4和CDK6的蛋白表达水平;MTT比色法、平板克隆形成实验检测干扰TLR4表达后肝癌细胞增殖能力.结果:在乙型肝炎相关性肝癌患者组织中,TLR4、CDK4和CDK6蛋白的总体表达水平高于相应癌旁组织(P<0.05),且TLR4分别与CDK4、CDK6的表达呈正相关性(r_1=0.66,r_2=0.57).成功构建并筛选出最佳干扰序列TLR4-siRNA-03.转染TLR4-siRNA-03后,与Control组相比,TLR4-siRNA组中CDK4、CDK6蛋白表达均随之降低(P<0.05),肝癌细胞Hep-3B的生长速度也明显下降(P<0.05),TLR4-siRNA组克隆形成率(18.77%±4.61%)也明显下降(P<0.05).结论:在乙型肝炎相关性肝癌中TLR4可能通过调控CDK4、CDK6来影响其增殖.AIM：To explore the effect of Toll-like receptor4（TLR4） on the proliferation of hepatitis B virus（HBV） related hepatic carcinoma cells and the underlying mechanism.METHODS：The expression of TLR4,cyclindependent kinase（CDK） 4 and CDK6 protein in 36 HBV-related hepatic carcinoma tissues and matched adjacent tissues were detected by Western blot,and their correlations in carcinoma tissues were analyzed.Three TLR4 specific siRNAs and a negative control were synthesized and transfected into hepatoma cells Hep-3B using liposomes.The siRNA sequence with the best performance was selected for Western blot analysis.The expression of TLR4,CDK4 and CDK6 proteins was measured by Western blot assay before and after transfection.The proliferation of hepatoma cells was observed by MTT assay and colony formation assay.RESULTS：The overall expression levels of TLR4,CDK4 and CDK6 proteins in HBVrelated hepatic carcinoma were significantly higher than those in the matched adjacent tissues（P 0.05）.There was a positive correlation between TLR4 and CDK4 expression（r = 0.66,P 0.05）,and between TLR4 and CDK6 expression（r = 0.57,P 0.05）.Using the best interference sequence（TLR4-siRNA-03）,it was found that the protein levels of CDK4 and CDK6 were significantly decreased in the TLR4-siRNA group（P 0.05）,compared to the control group.In addition,the proliferation of Hep-3B cells and the colony formation rate were both decreased（P 0.05） in the TLR4-siRNA group.CONCLUSION：TLR4 may regulate the proliferation of HBV-related hepatic carcinoma cells by controlling the expression of CDK4 and CDK6.

关 键 词：TOLL样受体4 周期蛋白依赖激酶4 周期蛋白依赖激酶6 乙型肝炎相关性肝癌 增殖 

分 类 号：R512.62[医药卫生—内科学] R735.7[医药卫生—临床医学]