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中华肝脏外科手术学电子杂志

中华肝脏外科手术学电子杂志 ›› 2014, Vol. 03 ›› Issue (05) : 312 -316. doi: 10.3877/cma.j.issn.2095-3232.2014.05.013

所属专题： 文献；

基础研究

miR-214对人肝细胞癌增殖能力影响的体外实验研究
郭明明1, 蔡锐彬1, 邹德志1, 赵坤1, 刘江辉1,†()   
  1. 1. 510080　广州，中山大学附属第一医院急诊科
  • 收稿日期:2014-06-16 出版日期:2014-10-10
  • 通信作者: 刘江辉
  • 基金资助:
    国家自然科学基金(81302550); 广东省自然科学基金(S2012040006483)

In vitro experimental study on impacts of miR-214 on the proliferation of human hepatocellular carcinoma

Mingming Guo1, Ruibin Cai1, Dezhi Zou1, Kun Zhao1, Jianghui Liu1,†()   

  1. 1. Emergency Department, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
  • Received:2014-06-16 Published:2014-10-10
  • Corresponding author: Jianghui Liu
  • About author:
    Corresponding author: Liu Jianghui, Email:
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引用本文：

郭明明, 蔡锐彬, 邹德志, 赵坤, 刘江辉. miR-214对人肝细胞癌增殖能力影响的体外实验研究[J]. 中华肝脏外科手术学电子杂志, 2014, 03(05): 312-316.

Mingming Guo, Ruibin Cai, Dezhi Zou, Kun Zhao, Jianghui Liu. In vitro experimental study on impacts of miR-214 on the proliferation of human hepatocellular carcinoma[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2014, 03(05): 312-316.

目的

探讨微小RNA（miR）-214对肝细胞癌（肝癌）增殖能力的影响及其相关机制。

方法

应用终浓度50 nmol/L的miR-214模拟物mimics及其阴性对照mimics分别转染MHCC97L肝癌细胞建立M214组、阴性对照组（NC214组），另设未转染对照组（Ctrl组）。采用荧光定量聚合酶链反应（PCR）检测3组肝癌细胞miR-214含量。采用细胞计数试剂盒（CCK）-8法检测细胞增殖抑制率，平板克隆形成实验检测细胞克隆形成能力，流式细胞术检测细胞凋亡率，蛋白质印迹法（Western blot）检测c-myc、Bax、B细胞淋巴瘤（Bcl）-2蛋白表达。3组比较采用单因素方差分析，两两比较采用LSD-t检验。

结果

M214组MHCC97L细胞miR-214的含量为Ctrl组的（2 536±7）倍，差异有统计学意义（LSD-t=58.75，P<0.05）。M214组24、48、72 h时细胞增殖抑制率分别为（15.33±0.62）%、（24.07±0.75）%和（41.02±0.91）%，与Ctrl组比较差异有统计学意义(LSD-t =14.64，19.87，31.86；P<0.05)。M214组细胞平板克隆形成数量（5.3±0.7）个/孔，明显低于Ctrl组的（37.1±1.0）个/孔(LSD-t =-12.51，P<0.05)。M214组细胞凋亡率（42.1±3.2）%明显高于Ctrl组的（7.0±0.7）% (LSD-t=35.66，P<0.05)。M214组c-myc、Bcl-2蛋白表达量降低，Bax蛋白表达量升高。

结论

miR-214可能通过下调c-myc蛋白表达及Bcl-2/Bax比值抑制人肝癌细胞的增殖能力。

关键词: 癌，肝细胞, 微RNAs, 细胞增殖, 细胞凋亡
Objective

To investigate the impact of micro ribonucleic acid -214 (miR-214) on the proliferation of hepatocellular carcinoma (HCC) and its mechanism.

Methods

MHCC97L cells were respectively transfected using miR-214 mimics and negative control mimics to establish M214 group, negative control (NC214) group. And untransfected control (Ctrl) group was established. The contents of miR-214 of MHCC97L cells in three groups were detected by fluorescence quantitative polymerase chain reaction (PCR). Cell counting kit (CCK)-8 assay was used to define the cell proliferation inhibition rate. Plate cloning formation assay was used to define the cell clonality. Cell apoptosis rate was detected by flow cytometery. The expressions of protein c-myc, Bax and B-cell lymphoma (Bcl)-2 were detected by Western blot assay. The comparison of three groups was conducted using one way analysis of variance and pairwise comparison using LSD-t test.

Results

The miR-214 content of MHCC97L cells in M214 group was (2 536±7) times of that in Ctrl group, where significant difference was observed (LSD-t=58.75, P<0.05). The cell proliferation inhibition rates were (15.33±0.62) %, (24.07±0.75) %, (41.02±0.91) % at the 24, 48, 72 h in M214 group, and significant difference was observed compared with that in Ctrl group (LSD-t =14.64, 19.87, 31.86; P<0.05). The clone formation quantity was (5.3±0.7) /well in M214 group, which was significantly lower compared with that in Ctrl group [(37.1±1.0)/well] (LSD-t =-12.51, P<0.05). The cell apoptosis rate was (42.1±3.2)% in M214 group, which was significantly higher compared with that in Ctrl group [(7.0±0.7) % ] (LSD-t=35.66, P<0.05). In M214 group, the expressions of protein c-myc, Bcl-2 decreased and Bax increased.

Conclusion

The miR-214 can inhibit the proliferation of human HCC cells through down-regulating the protein c-myc expression and Bcl-2/Bax ratio.

Key words: Carcinoma, hepatocellular, MicroRNAs, Cell proliferation, Apoptosis
图1 M214组、NC214组及Ctrl组MHCC97L细胞增殖抑制率的比较
图2 M214组、NC214组及Ctrl组MHCC97L细胞平板克隆形成情况
图3 Western blot法检测三组MHCC97L细胞c-myc、Bax、Bcl-2蛋白的表达
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