hMMS2基因对结肠癌细胞耐药逆转的影响
收稿日期: 2013-10-12
修回日期: 2014-01-29
网络出版日期: 2014-03-20
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
Received date: 2013-10-12
Revised date: 2014-01-29
Online published: 2014-03-20
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
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 . DOI: 10.3724/SP.J.1005.2014.0346
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.
[1] 王郭虹, 杨鸿, 姜凯, 张斌, 申乐. 新辅助化疗对结肠癌患者手术及预后的影响. 中国医药导刊, 2013, 15(6): 1012–1013. <\p>
[2] Shekhar MP. Drug resistance: challenges to effective therapy. Curr Cancer Drug Targets, 2011, 11(5): 613–623. <\p>
[3] 张舒羽, 王慧博, 卢大儒. 跨损伤DNA合成通路在肿瘤发生中的作用及其与化疗敏感性的关系. 癌变畸变突变, 2009, 21(3): 243–245. <\p>
[4] 李利坚, 隋御, 周翔, 王婷, 张蕾, 李元杰, 徐方. 干扰REV3L基因表达逆转结肠癌的耐药性. 基础医学与临床, 2013, 33(5): 542–547. <\p>
[5] 陈建明, 余应年, 陈星若. 反义阻断hMMS2基因表达以抑制细胞生长. 中国药理学与毒理学杂志, 2000, 14(3): 216–221. <\p>
[6] Broomfield S, Chow BL, Xiao W. MMS2, encoding a ubiquitin–conjugating–enzyme–like protein, is a member of the yeast error–free postreplication repair pathway. Proc Natl Acad Sci USA, 1998, 95(10): 5678–5683. <\p>
[7] Albertella MR, Green CM, Lehmann AR, O’Connor MJ. A role for polymerase eta in the cellular tolerance to cisplatin– induced damage. Cancer Res, 2005, 65(21): 9799–9806. <\p>
[8] Doles J, Oliver TG, Cameron ER, Hsu G, Jacks T, Walker GC, Hemann MT. Suppression of REV3, the catalytic subunit of Polζ, sensitizes drug–resistant lung tumors to chemotherapy. Proc Natl Acad Sci USA, 2010, 107(48): 20786–20791. <\p>
[9] Lin XJ, Trang J, Okuda T, Howell SB. DNA polymerase zeta accounts for the reduced cytotoxicity and enhanced mutagenicity of cisplatin in human colon carcinoma cells that have lost DNA mismatch repair. Clin Cancer Res, 2006, 12(2): 563–568. <\p>
[10] Okuda T, Lin XJ, Trang J, Howell SB. Suppression of hREV1 expression reduces the rate at which human ovar-ian carcinoma cells acquire resistance to cisplatin. Mol Pharmacol, 2005, 67(6): 1852–1860. <\p>
[11] Yuan BB, Xu Y, Woo JH, Wang YY, Bae YK, Yoon DS, Wersto RP, Tully E, Wilsbach K, Gabrielson E. Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability. Clin Cancer Res, 2006, 12(2): 405–410. <\p>
[12] Shen X, Jun S, O’Neal LE, Sonoda E, Bemark M, Sale JE, Li L. REV3 and REV1 play major roles in recombination– independent repair of DNA interstrand cross– links medi-ated by monoubiquitinated proliferating cell nuclear anti-gen (PCNA). J Biol Chem, 2006, 281(20): 13869–13872. <\p>
[13] Hicks JK, Chute CL, Paulsen MT, Ragland RL, Howlett NG, Gueranger Q, Glover TW, Canman CE. Differential roles for DNA polymerases eta, zeta, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross–links. Mol Cell Biol, 2010, 30(5): 1217–1230. <\p>
[14] Sharma S, Shah NA, Joiner AM, Roberts KH, Canman CE. DNA Polymerase ζ is a major determinant of resistance to platinum–based chemotherapeutic agents. Mol Pharmacol, 2012, 81(6): 778–787. http://molpharm.aspetjournals.org/content/81/6/778.full - fn-1#fn-1 <\p>
[15] Kim H, D’Andrea AD. Regulation of DNA cross–link re-pair by the Fanconi anemia/BRCA pathway. Genes Dev, 2012, 26(13): 1393–1408. <\p>
[16] Coulon S, Ramasubramanyan S, Alies C, Philippin G, Lehmann A, Fuchs RP. Rad8Rad5/Mms2–Ubc13 ubiquitin ligase complex controls translesion synthesis in fission yeast. EMBO J, 2010, 29(12): 2048–2058. <\p>
[17] Andersen PL, Xu F, Ziola B, McGregor WG, Xiao W. Se-quential assembly of translesion DNA polymerases at UV–induced DNA damage sites. Mol Biol Cell, 2011, 22(13): 2373–2383. <\p>
[18] Knobel PA, Kotov IN, Felley–Bosco E, Stahel RA, Marti TM. Inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. Neoplasia, 2011, 13(10): 961–970. <\p>
[19] He G, Kuang J, Koomen J, Kobayashi R, Khokhar AR, Siddik ZH. Recruitment of trimeric proliferating cell nu-clear antigen by G1–phase cyclin–dependent kinases fol-lo
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
