摘要
目的:探讨miR-195/Toll样受体4(TLR4)分子轴通过调控NF-κB通路对肝癌细胞增殖、侵袭和迁移的影响。方法:收集2016年3月至2017年1月昆明医科大学第二附属医院外科手术切除的25例肝癌组织以及对应的癌旁组织标本;肝癌HepG2细胞培养完成后分为4组,即对照组(NC)、miR-195 mimic组(miR-195组)、TLR4敲降组(si-TLR4组)和miR-195 inhibitor联合TLR4敲降组(si-TLR4+miR-195 inhibitor组)。采用q PCR检测miR-195在肝癌组织和细胞系中的表达水平;采用CCK-8法检测上述各组细胞的增殖活力,Transwell法检测各组细胞的侵袭能力,划痕愈合实验检测细胞迁移能力,双荧光素酶报告基因验证miR-195和TLR4的靶向调控关系,WB检测TLR4和NF-κB p65蛋白的表达。结果:miR-195在肝癌组织中低表达(P<0.01)。相比于人肝上皮细胞(THLE-3),miR-193在肝癌细胞系(HepG2和Huh-7)中低表达(P<0.01),且HepG2细胞中的表达水平最低。过表达miR-195后HepG2细胞增殖活力明显低于对照组(P<0.01),穿膜细胞明显减少(P<0.01),HepG2细胞迁移能力明显下调(P<0.01)。过表达miR-195可明显抑制TLR4蛋白的表达水平(P<0.05),且TLR4与miR-195的表达呈负相关(R^2=0.602,P<0.0001)。过表达miR-195可靶向下调TLR4并阻断NF-κB通路抑制HepG2细胞增殖、侵袭和迁移能力(P<0.05或P<0.01)。结论:miR-195能够抑制HepG2细胞增殖、侵袭及迁移能力,其机制可能与靶向调控TLR4并阻断NF-κB通路影响细胞生物学行为有关。
Objective:To explore the effect of miR-195/TLR4 axis on the proliferation,invasion and migration of liver cancer cells via regulating NF-κB pathway.Methods:Twenty-five pairs of liver cancer tissues and corresponding adjacent tissues surgically resected at the Second Affiliated Hospital of Kunming Medical University from March 2016 to January 2017 were collected for this study.Liver cancer HepG2 cells were cultured and then randomly divided into four groups:control group(NC),miR-195 mimic group(miR-195),TLR4 knockdown group(si-TLR4),and miR-195 inhibitor combined with TRL4 knockdown group(si-TLR4+miR-195 inhibitor).qRTPCR was used to detect the expression of miR-195 in liver cancer tissues and cell lines.CCK-8 assay was used to evaluate the cell viability of each group.Transwell and Wound healing assay were applied to detect the invasion and migration ability of HepG2 cells,respectively.Dual-luciferase reporter gene assay was used to verify the targeted regulation of TLR4 by miR-195.WB was applied to analyze the protein expressions of TLR4 and NF-κB p65.Results:miR-195 was down-regulated in the liver cancer tissues compared with adjacent tissues(P<0.01).Compared with human hepatic epithelial cells(THLE-3),the expression of miR-193 in liver cancer cell lines(HepG2 and Huh-7)was down-regulated(P<0.01),and the expression level in HepG2 cells was the lowest.The proliferation,invasion and migration of HepG2 cells was significantly suppressed after over-expression of miR-195(all P<0.01).Moreover,over-expression of miR-195 significantly down-regulated TLR4 protein expression(P<0.05),and TLR4 was negatively correlated with miR-195(R^2=0.602,P<0.0001).Furthermore,miR-195 over-expression inhibited proliferation,invasion and migration of HepG2 cells by targeting TLR4 expression and blocking NF-κB pathway(P<0.05 or P<0.01).Conclusion:miR-195 over-expression can inhibit the proliferation,invasion and migration of HepG2 cells.The mechanism may be related with targeting TLR4 and blocking the NF-κB pathway to affect cell biol
作者
陈鹏
崔锐
梁鸿飞
陈晨
CHEN Peng;CUI Rui;LIANG Hongfei;CHEN Chen(Department of Hepatic-Biliary-Pancreatic Surgery,The Second Affiliated Hospital of Kunming Medical University,Kunming 650101,Yunnan,China;Department of Ophthalmology,The Second People's Hospital of Yunnan Province,Kunming 650021,Yunnan,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2019年第5期550-556,共7页
Chinese Journal of Cancer Biotherapy
基金
云南省科技计划项目-面上项目(No.2014FB194
2016FB133)
云南省应用基础研究(昆明医科大学联合专项)[No.2017FE467(-182)]~~
