Zhang Lei, Sui Yu, Wang Ting, Li Lijian, Li Yuanjie, Jin Caixia, Xu Fang
Ningxia Key Laboratory of Reproduction and Genetics, College of Inspection, Ningxia Medical University, Yinchuan 750004, China.
Center for Translational Medicine, School of Medicine, Tongji University, Shanghai 200072, China.
Yi Chuan. 2014 Apr;36(4):346-53.
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.
在本研究中，通过使用高分化人结肠直肠癌L-OHP耐药细胞系THC8307/L-OHP，研究了编码人泛素结合酶E2变体2的hMMS2（人甲磺酸甲酯敏感突变体2）基因在人结肠癌耐药性中的作用。将THC8307/L-OHP细胞通过脂质体与表达针对hMMS2的miRNA和GFP的质粒pcDNA6.2-GW/EmGFP-miR-MMS2一起转染，随后进行实时荧光定量PCR和免疫荧光以选择hMMS2表达显著降低的稳定转染子。与未转染或pcDNA6.2-GW/EmGFP载体转染的细胞相比，hMMS2缺失的细胞在用奥沙利铂（L-OHP）处理后，半数抑制浓度（IC50）、耐药指数（RI）和集落形成效率（CFE）显著降低（P<0.05），而其相对逆转效率（RRE）显著高于对照细胞（P<0.05），表明细胞增殖能力受损。事实上，罗丹明123染色和流式细胞术分析显示hMMS2缺失的细胞凋亡率增加，而两种对照细胞系之间未观察到细胞增殖或凋亡的差异。上述观察结果共同表明，抑制hMMS2可通过促进凋亡逆转分化型人结肠癌细胞对L-OHP的耐受性。
