hMMS2基因对结肠癌细胞耐药逆转的影响

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。

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

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.