实用肝脏病杂志 ›› 2018, Vol. 21 ›› Issue (5): 701-704.doi: 10.3969/j.issn.1672-5069.2018.05.012

• 实验性肝炎 • 上一篇    下一篇

let-7a调控自噬对缺氧状态下原发性肝癌HCCLM3细胞增殖的影响*

佟辉, 李涛, 申川, 彭承宏, 邱伟华, 沈柏用, 祝哲诚   

  1. 200025 上海市 上海交通大学医学院附属瑞金医院普外科
  • 收稿日期:2017-07-28 出版日期:2018-09-10 发布日期:2018-09-27
  • 通讯作者: 祝哲诚,E-mail:zhuzhecheng@126.com
  • 作者简介:佟辉,男,31 岁,医学硕士。研究方向:肝细胞癌防治基础与临床研究。E-mail:drtonghui@163.com

Effect of let-7a on proliferation of HCCLM3 cells under hypoxia by regulating autophagy in vitro

Tong Hui, Li Tao, Shen Chuan, et al   

  1. Department of General Surgery,Ruijin Hospital,Jiaotong University School of Medicine,Shanghai 200025
  • Received:2017-07-28 Online:2018-09-10 Published:2018-09-27

摘要: 目的 探讨let-7a调控自噬对缺氧状态下原发性肝癌HCCLM3细胞增殖的影响。方法 将HCCLM3细胞分为常氧组、缺氧组和缺氧let-7a组,分别给予不同的处理。各组设6个平行孔,培养72 h。采用吖啶橙染液法测定HCCLM3细胞自噬率,采用MTT法检测HCCLM3细胞活力,使用流式细胞仪分析HCCLM3细胞周期G1,采用酶联免疫吸附法测定培养上清let-7a、细胞周期D1蛋白(cyc D1)和血管内皮生长因子(VEGF)水平。结果 缺氧let-7a组细胞存活率为(49.7±9.5)%,显著低于常氧组或缺氧组的(100.0±0.0)%或(119.1±13.1)%(P<0.05),缺氧组细胞存活率显著高于常氧组(P<0.05);缺氧let-7a组细胞周期G1期和细胞自噬率分别为(89.6±16.1)%和(49.3±7.4)%,显著高于常氧组或缺氧组的(56.3±11.0)%和(13.2±8.1)%或(69.7±14.1)%和(23.5±5.5)%(P<0.05),缺氧组细胞周期G1期和自噬率显著高于常氧组(P<0.05);缺氧let-7a组细胞let-7a蛋白水平为(96.1±9.3) μmol/L,显著高于常氧组或缺氧组的(33.4±7.1)μmol/L或(65.3±5.2)μmol/L(P<0.05),cyc D1和VEGF水平分别为(72.3±8.8)μmol/L和(56.3±6.3)μmol/L,显著低于常氧组或缺氧组的(124.1±7.2)μmol/L和(114.1±5.2) μmol/L或(98.2±6.7) μmol/L和(85.2±8.1)μmol/L(P<0.05),缺氧组let-7a蛋白显著低于常氧组(P<0.05),cyc D1和VEGF显著高于常氧组(P<0.05)。结论 上调let-7a在肝癌HCCLM3细胞的表达能诱发细胞自噬,使得细胞在G1期往S期的分化过程发生障碍,抑制细胞在缺氧环境中的增殖,其机制可能与细胞CYC D1和VEGF表达下调有关。

关键词: 肝细胞癌, HCCLM3细胞, let-7a, 缺氧, 细胞自噬

Abstract: Objective To investigate the effect of let-7a on proliferation of HCCLM3 cells under hypoxia by regulating autophagy in vitro. Methods The HCCLM3 cells from human hepatocellular carcinoma were divided into three groups,e.g. the normoxia group:5×106/mL HCCLM3 cells in 10% fetal bovine DMEM medium were cultured in 37 ℃,5% CO2,20% O2,hypoxia group:in 37 ℃,5% CO2,2% O2,93%N2 and hypoxia let-7a group:in 37 ℃,5% CO2,2% O2,93%N2. Six parallel samples were prepared in each group and cultured for 72 h. After incubation,the autophagy of HCCLM3 cells was detected by acridine orange staining,the activity of HCCLM3 cells was detected by MTT,the HCCLM3 cell cycle analysis G1 was detected by flow cytometry,and the let-7a,cyc D1 and vascular endothelial growth factor (VEGF) levels in supernatant were determined by ELISA. Results The cell survival rate in oxygen let -7 group was(49.7±9.5) %,significantly lower than in normal oxygen or hypoxia group [(100.0±0.0) % or (119.1±13.1) %,P<0.05],the cell survival rate in hypoxia group was significantly higher than that in the normal oxygen group (P<0.05);the cell cycle G1 phase and the rate of autophagy in oxygen let -7a group were (89.6±16.1) % and (49.3±7.4) %,significantly higher than those in normal oxygen or hypoxia group [(56.3±11.0)% and(13.2±8.1)% or(69.7±14.1)% and(23.5±5.5)%(P<0.05);the let-7a level in the supernatant in the oxygen let -7a group was(96.1±9.3) μmol/L,much higher than in the normal oxygen or hypoxia group [(33.4±7.1)μmol/L or (65.3±5.2)μmol/L,P<0.05],cyc D1 and VEGF levels were(72.3±8.8)μmol/L and(56.3±6.3)μmol/L,much lower than in the normal oxygen or hypoxia group [(124.1±7.2)μmol/L and(114.1±5.2) μmol/L or (98.2±6.7) μmol/L and (85.2±8.1)μmol/L,P<0.05]. Conclusion The upregulation of let-7a expression in hepatocellular carcinoma HCCLM3 cells in vitro can induce cell autophagy,make the transformation disorder of the cells in G1 phase to S phase,inhibit the proliferation of cells in hypoxia,and the mechanism might be the decreased CYC D1 and VEGF expression.

Key words: Hepatocellular carcinoma, HCCLM3 cells, Let-7a, Autophagy, Hypoxia.