miR-155对人肺癌95D细胞生物学行为的影响  

Biological effects of miR-155 on human lung cancer 95D cell

作　　者：赵娟娟[1] 李永菊[1] 陈超[1] 郭萌萌[1] 陶弋婧 任涛[2] 徐林[1] 

机构地区：[1]遵义医学院免疫学教研室,贵州遵义563000 [2]同济大学附属上海东方医院呼吸内科,上海200120

出　　处：《中国肿瘤生物治疗杂志》2014年第5期510-515,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81260398;No.31370918);教育部新世纪优秀人才计划(No.NCET-12-0661)~~

摘　　要：目的:构建携microRNA-155(miR-155)的真核表达载体并观察其转染高转移性人巨细胞肺癌95D细胞后细胞的生物学行为变化。方法:以95D细胞基因组RNA为模板,经PCR法扩增miR-155的前体序列,由BamHⅠ和HindⅢ双酶切后将其亚克隆入真核表达载体pcDNA 3.1(-),并进行双酶切及测序鉴定;将构建成功的pcDNA3.1(-)-pri-miR-155载体(命名为p-miR-155)体外瞬时转染人肺癌95D细胞,利用Real-time PCR探针法检测miR-155成熟体的表达水平,并利用CCK-8法、克隆形成实验和划痕法检测95D细胞的增殖、克隆形成以及体外迁移能力。结果:成功构建携miR-155的真核表达载体;与空白(Mock)和对照组(p-Ctrl)相比,转染后的95D细胞过表达miR-155[(2.04±0.62)vs(0.76±0.62)、(1.00±0.45),均P<0.01]、p-miR-155载体转染组95D细胞的增殖抑制明显增加[(46.70±6.89)%vs(3.70±1.40)%、(1.11±0.75)%,P<0.01]、克隆形成能力(在100和1 000个细胞/孔接种条件下)明显下降[(12±3)vs(34±3)、(35±3)个,P<0.01;(78±4)vs(159±4)、(165±4)个,P<0.01)],此外,细胞的迁移细胞数也明显减少[(110±5)vs(295±5)、(325±5)个,P<0.01]。结论:通过miR-155真核表达载体转染产生的过表达miR-155可显著抑制人肺癌95D细胞的增殖和迁移能力。Objective： To evaluate the effect of microRNA-155（ miR-155） on human lung cancer cell behaviors in vitro. Methods： A plasmid vector（ p-miR-155） carrying the pri-miR-155 sequence amplified from genomic RNA of human lung cancer 95 D cells by PCR was constructed. Lung cancer 95 D cells were transiently transfected with p-miR-155. p-miR-155 mRNA abundance in 95 D cells was assessed by real-time PCR before and after transfection. Cell proliferation and migration in control,mock-transfected and transfected 95 D cells were assessed by CCK-8 assay wound assay respectively. Results： The abundance of miR-155 mRNA was increased significantly in 95 D cells transfected with p-miR-155 than in mock-transfected and control cells（ 2. 045 ± 0. 62 vs 0. 76 ± 0. 62,1 ± 0. 45; P〈 0. 01）. The proliferation was markedly inhibited in p-miR-155 transfectants as compared in mock-transfected and control cells（ [46. 70 ± 6. 89]%vs [3. 70 ± 1. 40]%,[1. 11 ± 0. 75]%; P 〈0. 01）. The number of colonies formed was significantly decreased（ [12 ±3]vs [34 ± 3],[35 ± 3]; P〈 0. 01） and so was migration capability（ [110 ± 5]vs [295 ± 5],[325 ± 5]; P 〈0. 01）in p-miR-155-transfected cells as compared with mock-transfected and control cells. Conclusion： A eukaryotic expression vector carrying human pri-miR-155 sequence is capable of effectively inhibiting lung cancer cell proliferation and migra-tion,thus having a significant clinical implication.

关 键 词：microRNA-155(miR-155) 真核表达 肺癌 95D细胞 增殖 迁移 

分 类 号：R734.2[医药卫生—肿瘤] R730.54[医药卫生—临床医学]