hMMS2基因对结肠癌细胞耐药逆转的影响

doi:10.3724/SP.J.1005.2014.0346

遗传 ›› 2014, Vol. 36 ›› Issue (4): 346-353.doi: 10.3724/SP.J.1005.2014.0346

hMMS2基因对结肠癌细胞耐药逆转的影响

张蕾¹,隋御²,王婷¹,李利坚¹,李元杰¹,金彩霞²,徐方¹

1. 宁夏医科大学检验学院, 宁夏生殖与遗传重点实验室, 银川 750004;
2. 同济大学医学院转化医学研究中心, 上海 200072

收稿日期:2013-10-12 修回日期:2014-01-29 出版日期:2014-04-20 发布日期:2014-03-20
通讯作者: 徐方, 教授, 博士生导师, 研究方向：DNA损伤与修复。E-mail: xufang@nxmu.edu.cn E-mail:xufang@nxmu.edu.cn
作者简介:张蕾, 在读硕士研究生, 专业方向：DNA损伤与修复。E-mail: 2427084464@qq.com
基金资助:
国家自然科学基金项目(编号：81060170, 31360251); 教育部“春晖计划”项目(编号：Z2011056); 银川市应用研究开发计划项目(编号：银财发(2012)249)资助

Roles of hMMS2 gene in reversing the oxaliplatin tolerance of hu-man colon carcinoma cells

Lei Zhang¹, Yu Sui², Ting Wang³, Lijian Li¹, Yuanjie Li⁴, Caixia Jin², Fang Xu¹

1. Ningxia Key Laboratory of Reproduction and Genetics, College of Inspection, Ningxia Medical University, Yinchuan 750004, China;
2. Center for Translational Medicine, School of Medicine, Tongji University, Shanghai 200072, China

Received:2013-10-12 Revised:2014-01-29 Online:2014-04-20 Published:2014-03-20

摘要/Abstract

摘要：

为探讨hMMS2(Human methyl methanesulfonate sensitive mutant 2)基因对人结肠癌细胞耐药逆转的影响, 文章以人高分化耐奥沙利铂结肠癌细胞(THC8307/L-OHP)为实验材料, 采用脂质体-质粒转染技术构建了带有干扰目的基因hMMS2的miRNA片段并携带绿色荧光蛋白标记重组质粒(pcDNA6.2-GW/EmGFP-miR-MMS2)的细胞系, 通过实时荧光定量PCR(qRT-PCR)和免疫荧光技术(Immunostaining technique)检测该细胞系的干扰效率。选择hMMS2低表达具有统计学意义的上述细胞系作为实验组细胞, 同时将未曾作过处理的THC8307/ L-OHP细胞作为空白对照组, 转染绿色荧光蛋白空质粒(pcDNA6.2-GW/EmGFP-miR)的THC8307/L-OHP细胞作为阴性对照组, 以噻唑蓝比色分析实验(MTT colorimetric analysis assay)、克隆形成实验(Colony formation assay)对3组细胞的存活率和克隆形成率进行检测, 结果显示：实验组细胞的奥沙利铂半数抑制浓度(Half inhibition concentration, IC50)、耐药指数(Resistance index, RI)及克隆形成率(Colony-forming efficiency, CFE)均比对照组细胞明显降低(P＜0.05), 而相对逆转率(Relative reverse efficiency, RRE)增高(P＜0.05), 提示实验组细胞增殖能力减弱; 以罗丹明123实验(Rhodamine 123 assay)结合倒置荧光显微镜、流式细胞仪检测技术等观测细胞的凋亡变化, 结果显示, 实验组细胞的凋亡率较对照组细胞显著增高(P＜0.05); 两对照(空白、阴性)组间并无细胞增殖或凋亡的显著性差异。研究结果提示：下调hMMS2基因表达可逆转人高分化耐奥沙利铂结肠癌细胞对L-OHP的耐药性并促进结肠癌细胞的凋亡。

关键词: RNA干扰, 铂类耐药, hMMS2, 人高分化耐奥沙利铂结肠癌细胞(THC8307/L-OHP), 细胞凋亡

Abstract:

In this study, the roles of hMMS2 (human methyl methanesulfonate sensitive mutant 2) gene encoding the human ubiquitin-conjugating enzyme E2 variant 2 in the drug resistance in human colon carcinoma were investigated by using a well-differentiated human colorectal carcinoma L-OHP-resistant cell line, THC8307/L-OHP. THC8307/L-OHP cells were transfected via liposome along with plasmid pcDNA6.2-GW/EmGFP-miR-MMS2 expressing both miRNA against hMMS2 and GFP, followed by real-time fluorescent quantitative PCR and immunofluorescence to select stable transfectants with significantly reduced hMMS2 expression. Compared with untransfected or pcDNA6.2-GW/EmGFP vector-transfected cells, the hMMS2-depleted cells displayed significantly (P<0.05) reduced half inhibition concentration(IC50) resistance index (RI) and colony-forming efficiency (CFE) upon treatment with oxaliplatin (L-OHP), while its relative reverse efficiency(RRE) was significantly higher (P<0.05) than the control cells, indicating compromised ability of cell proliferation. Indeed, Rho-damine 123 staining and flow cytometry analyses revealed an increased rate of apoptosis in hMMS2-depleted cells while no difference in cell proliferation or apoptosis was observed between the two control cell lines. The above observations collec-tively indicate that suppression of hMMS2 reverses L-OHP tolerance in differentiated human colorectal carcinoma cells by promoting apoptosis.

Key words: RNA interference, platinum resistance, hMMS2, human well-differentiated colorectal carcinoma L-OHP-resistant cell line, apoptosis

张蕾, 隋御, 王婷, 李利坚, 李元杰, 金彩霞, 徐方. hMMS2基因对结肠癌细胞耐药逆转的影响[J]. 遗传, 2014, 36(4): 346-353.

Lei Zhang, Yu Sui, Ting Wang, Lijian Li, Yuanjie Li, Caixia Jin, Fang Xu. Roles of hMMS2 gene in reversing the oxaliplatin tolerance of hu-man colon carcinoma cells[J]. HEREDITAS, 2014, 36(4): 346-353.

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hMMS2基因对结肠癌细胞耐药逆转的影响

Roles of hMMS2 gene in reversing the oxaliplatin tolerance of hu-man colon carcinoma cells

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