RNA干扰NUP88基因对人乳腺癌MCF-7细胞生长及侵袭力的影响  

Effect of Silencing Nup88 Gene by RNA Interference on Growth and Invasion in Human Breast Cancer MCF-7 Cell

作　　者：李玉强[1] 朱志图[1] 王巍[1] 李谌[1] 徐娜[1] 王钰[1] 李男[1] 孙宏治[1] 

机构地区：[1]辽宁医学院附属第一医院,锦州121001

出　　处：《中国生物工程杂志》2014年第9期31-39,共9页China Biotechnology

基　　金：辽宁省教育厅教学计划(L201145);辽宁省高等学校优秀人才支持计划(LJQ2011091)资助项目

摘　　要：目的:构建重组慢病毒介导的NUP88-shRNA载体,通过RNAi技术分别观察沉默NUP88后对MCF-7增殖,粘附,侵袭和转移情况的影响,为乳腺癌的临床基因治疗寻找新的靶点。方法:构建NUP88重组慢病毒表达载体,包装后检测滴度,以最佳复感染指数转染乳腺癌MCF-7细胞,利用RT-PCR和Western blot检测各组MCF-7细胞中mRNA和蛋白的表达效率;MTT法和流式细胞仪检测法,检测NUP88基因被干扰后对MCF-7细胞增殖和凋亡的影响;细胞侵袭实验检测NUP88基因被干扰后对MCF-7侵袭力的影响。结果四组病毒及一组阴性对照均构建成功,滴度均为4E+8TU/ml;RT-PCR和Western blot检测,结果表明:经NUP88-shRNA转染的MCF-7细胞组NUP88 mRNA和蛋白质的表达与经阴性转染组和空白MCF-7细胞组相比,差异明显具有统计学意义(P<0.01);测定NUP88-shRNA1组沉默效率最高,沉默率可达到86%;MTT法结果表明:实验组经NUP88-shRNA1慢病毒转染后细胞增殖程度显著减少,与空白组和对照组相比有显著性差异(P<0.05)。流式细胞仪检测三组MCF-7细胞凋亡结果表明:实验组经慢病毒转染后细胞凋亡率显著增加,与对照组和空白组相比有显著性差异(P<0.05);细胞侵袭实验表明:在肿瘤细胞常规培养24h后,实验组与空白组和阴性对照组比较,穿膜细胞数量明显减少,具有显著性差异(P<0.05)结论:NUP88重组慢病毒可以通过RNAi成功抑制MCF-7中NUP88基因的表达,并能显著抑制其增殖及远处的侵袭能力。Objective： Construct recombinant lentivirus NUP88 - shRNA mediated by the carrier, by RNAi technology respectively to observe silence after NUP88 MCF - 7 proliferation, adhesion, invasion and metastasis of influence, looking for new targets for clinical gene therapy of breast cancer. Methods Build NUP88 Lentivirus vectors, packaging test after drop, to MOI cell transfection breast cancer MCF-7, using RT-PCR and Western- blot test each mRNA and protein expression in MCF-7 cell efficiency; Determined by MTT method and flow cytometry assay, after testing NUP88 gene is interference effects on MCF-7 cell proliferation and apoptosis; Cell invasion after experimental detection NUP88 gene is interference effects on MCF-7 aggressivity. Results Four groups of virus and a negative control group are building a successful, drop degrees are 4E ＋ 8 Tu/mL; RT-RCR and Western-blot test, the results show that the NUP88-shRNA transfection of MCF-7 cell group NUP88 mRNA and protein expression and the negative transfection group and blank MCF - 7 cell group compared with significant difference statistically significant （ P 〈 0.01 ） ; Determination of NUP88-shRNA1 group of silence, the most efficient silence rate can reach 86% ; Determined by MTT method, the results show that the experimental group after NUP88-shRNA1 Lentivirns transfection cell proliferation significantly reduced, compared with the blank group and the control group with significant difference （ P 〈 0.05 ） ; Flow cytometry instrument detection of three groups of MCF - 7 cell apoptosis results show that the experimental group after lentivirus transfection cell apoptosis rate increased significantly, compared with the control group and blank group had significant difference （P 〈 0.05 ） ; Cell invasion experiment show that the tumor cells to conventional training after 24 h, compared with the blank group and the negative control group, experimental group in membrane cells decreased significantly, with significant difference （ P 〈 O. 05 ）. Conc

关 键 词：NUP88 人乳腺癌细胞MCF-7 RNAI 慢病毒载体 

分 类 号：Q78[生物学—分子生物学]