摘要
目的:探讨Mindbomb E3泛素蛋白连接酶2(MIB2)对HepG2肝癌细胞恶性生长的影响,并分析其可能机制。方法:以HepG2肝癌细胞为研究对象,首先筛选过表达MIB2稳定株,利用细胞软琼脂克隆实验观察集落形成情况;使用炎性细胞因子TNF-α和RNA病毒SEV处理MIB2过表达稳定株和阴性对照4 h,免疫印迹法检测Notch1的表达水平;同时收取细胞培养上清,ELISA检测IL-6及TNF-α的分泌情况。其次通过小干扰RNA(siRNA)转染HepG2细胞,48 h后通过免疫印迹法验证MIB2敲低情况;同样使用TNF-α和SEV处理4 h,收取细胞培养上清,ELISA检测IL-6及TNF-α的分泌情况。结果:过表达MIB2对HepG2细胞的克隆形成有明显的抑制作用;过表达MIB2导致HepG2细胞IL-6本底水平的分泌和TNF-α及SEV诱导的分泌均下降,在上述各条件下均未检测到TNF-α的分泌,Notch1表达水平无显著变化。MIB2敲低后,HepG2细胞IL-6本底水平的分泌和TNF-α及SEV诱导的分泌均增强,反向验证上述效应。结论:MIB2能够明显抑制肝癌细胞的恶性生长,该效应可能和IL-6的表达下降有关。
Objective:To investigate the effect of Mindbomb E3 ubiquitin ligase 2(MIB2)on the oncogenic growth of HepG2 hepatocellular carcinoma cells and its underlying mechanism.Methods:Firstly,HepG2 stable clone over-expressing MIB2 was selected and anchorage-independent growth was observed using soft agar cloning assays.MIB2-overexpressing HepG2 cells and negative control were treated with pro-inflammatory cytokine TNF-αand RNA virus SEV for 4 h.The expression of Notch1 was detected by Western blot.The secretion of IL-6 and TNF-αwas detected by ELISA.Secondly,HepG2 cells were transfected with small interfering RNAs(siRNAs).48 h later,MIB2 knockdown was verified by Western blot.Similarly,cells were treated with TNF-αand SEV for 4 h,and cell culture supernatants were harvested for ELISA to detect the secretion of IL-6 and TNF-α.Results:Overexpression of MIB2 in HepG2 cells significantly inhibited anchorage-independent growth.Overexpression of MIB2 inhibited basal and IL-6 or TNF-αinduced IL-6 secretion from HepG2 cells,whereas no TNF-αsecretion could be detected.Notch1 expression remained unchanged.By contrast,MIB2 knockdown augmented basal and IL-6 or TNF-αinduced IL-6 secretion from HepG2 cells.Conclusion:MIB2 can anchorage-independent growth of HepG2 cells.This effect may be related to the decreased expression of IL-6.
作者
徐哲刘健
沈少灵
偏丽丽
黄晓峰
林周
修冰水
XU Zhe;LIU Jian;SHEN Shao-Ling;PIAN Li-Li;HUANG Xiao-Feng;LIN Zhou;XIU Bing-Shui(Anhui Medical University,Hefei 230000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2018年第9期1289-1292,1297,共5页
Chinese Journal of Immunology
基金
本文受北京市自然科学基金(7174329、5174042)资助。
