上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (12): 1581-.doi: 10.3969/j.issn.1674-8115.2012.12.014

• 论著（基础研究） • 上一篇    下一篇

miRNA765高表达对胃癌耐药细胞株BGC-823/CDDP耐药性的影响

霍中华, 储著凌, 胡 君, 宋 博, 吕 盛   

  1. 中国人民解放军第四五四医院普外科, 南京 210002
  • 出版日期:2012-12-28 发布日期:2012-12-31
  • 作者简介:霍中华（1958—）, 主任医师, 博士；电子信箱: hzh05091@126.com。

Effects of miRNA765 overexpression on drug resistance of resistant gastric carcinoma cell line BGC-823/CDDP

HUO Zhong-hua, CHU Zhu-ling, HU Jun, SONG Bo, LÜ|Sheng   

  1. Department of General Surgery, 454th Hospital of PLA, Nanjing 210002, China
  • Online:2012-12-28 Published:2012-12-31

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摘要：

目的 观察miRNA765高表达对胃癌顺铂（CDDP）耐药细胞株BGC-823/CDDP耐药性的影响。方法 将PCR扩增的包含miRNA765前体的基因序列克隆至真核表达载体，构建miRNA重组表达载体；阳离子脂质体转染重组质粒到BGC-823/CDDP细胞。转染后48 h，Real-Time PCR和Western blotting法分别检测细胞中miRNA765和细胞因子诱导的凋亡抑制因子1（CIAPIN1）蛋白的相对表达量；采用不同浓度CDDP处理转染后48 h的BGC-823/CDDP细胞，24和48 h后MTT法检测细胞活性并计算细胞对于CDDP的半抑制浓度（IC50）。采用终质量浓度为1 μg/mL的CDDP处理基因干预后的BGC-823/CDDP细胞，双染法检测细胞凋亡率。结果 BGC-823/CDDP细胞内miRNA765的相对表达量明显低于其亲本细胞BGC-823，CIAPIN1蛋白相对表达量则明显高于其亲本细胞株（均P<0.01）。在BGC-823/CDDP细胞内高表达miRNA765可明显抑制CIAPIN1蛋白的表达，转染后48 h的CIAPIN1蛋白表达量明显低于未转染组（P<0.01）；miRNA765高表达可明显降低BGC-823/CDDP细胞的耐药能力，48 h后IC50值从（18.27±3.92）μg/mL降至（1.50±0.43）μg/mL（P<0.05）。转染48 h后，BGC-823/CDDP细胞的凋亡率从（10.1±1.7）%上升至（53.4±7.9）%（P<0.01）。结论 miRNA765高表达可以明显降低胃癌耐药细胞株BGC-823/CDDP的耐药能力。

关键词: miRNA765, BGC-823/CDDP, 细胞因子诱导的凋亡抑制因子1, 肿瘤细胞耐药性, 顺铂

Abstract:

Objective To investigate the effects of miRNA overexpression on drug resistance of cis-diamminedichloroplatinum (CDDP) resistant gastric cancer cell line BGC-823/CDDP. Methods Human genomic DNA was extracted, PCR primers targeting human miRNA765 were designed, and gene sequence containing miRNA765 precursor was amplified by PCR and cloned into an eukaryotic expression vector to construct a recombinant miRNA expression vector. BGC823/CDDP cells were transfected with the recombinant plasmid using cationic liposome. Forty-eight hours after transfection, the relative contents of miRNA765 and cytokine-induced apoptosis inhibitor 1 (CIAPIN1) protein in cells were determined by Real-Time PCR and Western blotting. Forty-eight hours after transfection, the cells were treated with different concentrations of CDDP. Twenty-four and forty-eight hours later, cell viability was detected using MTT assay, and 50% concentration of inhibition (IC50) of the cells to CDDP was calculated. BGC-823/CDDP cells of gene intervention were treated with CDDP at a final concentration of 1 μg/mL, and double staining method was used to detect the cell apoptosis rate. Results The relative expression of miRNA765 in BGC-823/CDDP cells was significantly lower than that in parental cell line BGC-823 (P<0.01), and the relative expression of CIAPIN1 protein in BGC823/CDDP cells was significantly higher than that of parental cell line (P<0.01). The overexpression of miRNA765 significantly inhibited the expression of CIAPIN1 protein in BGC-823/CDDP cells. Forty-eight hours after transfection, the expression of CIAPIN1 protein was significantly lower than that of the untransfected group (P<0.01). The overexpression of miRNA765 significantly reduced the drug resistance of BGC-823/CDDP cells, and 48-hour IC50 value was reduced from (18.27±3.92) μg/mL to (1.50±0.43) μg/mL (P<0.05). Forty-eight hours after transfection, the apoptosis rate of BGC-823/CDDP cells was significantly increased from (10.1±1.7)% to (53.4±7.9)%（P＜0.01）. Conclusion The overexpression of miRNA765 may significantly reduce the drug resistance of resistant gastric carcinoma BGC-823/CDDP cells.

Key words: miRNA765, BGC-823/CDDP, cytokine-induced apoptosis inhibitor 1, drug resistance of tumor cells, cis-diamminedichloroplatinum