DAL-1对非小细胞肺癌细胞侵袭和迁移能力影响的初步研究  

Primary survey of effects of DAL-1 in procession of proliferation,migration and invasion of NSCLC cell line

作　　者：徐若冰[1] 张雅洁[1] 郭爱林[2] 

机构地区：[1]广州医学院病理学教研室,广东广州510182 [2]广东省人民医院医学研究中心生物芯片部,广东广州510060

出　　处：《中华肿瘤防治杂志》2009年第15期1139-1143,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省自然科学基金(7003068)

摘　　要：目的:通过RNAi技术建立DAL-1稳定抑制的NSCLC细胞实验模型,并对DAL-1在NSCLC细胞增殖、迁移和侵袭过程中的作用进行研究。方法:构建靶向DAL-1的shRNA表达载体转染NSCLC细胞株,筛选出阳性克隆,在mRNA和蛋白质水平鉴定其抑制率,最终得到DAL-1稳定抑制的细胞株;用Transwell迁移和侵袭模型比较实验和对照组细胞在迁移和侵袭能力方面的差异;用MTT法比较实验和对照组细胞在增殖能力方面的差异。结果:自行构建的pGPU6/GFP/Neo-DAL-1shRNA表达载体经双酶切鉴定和测序鉴定序列及插入方向正确,其中pGPU6/GFP/Neo-DAL-1-T4表达载体在mRNA和蛋白质水平能有效抑制DAL-1的表达,抑制率分别为(87.41±1.994)%和(82.73±2.147)%。与对照组细胞比较,DAL-1稳定抑制的实验组细胞迁移和侵袭能力(P=0.000)和增殖能力显著增强(P=0.000)。结论:成功设计并构建了靶向DAL-1的shRNA表达载体,建立了DAL-1稳定抑制的NSCLC细胞株,为进一步研究DAL-1在NSCLC细胞中的作用提供了实验模型。OBJECTIVE： To apply primary survey of effects of DAL-1 in procession of proliferation, migration and invasion of NSCLC cell line by construction of experimental NSCLC cellular model which DAL-1 is steady inhibited. METHODS： To design and construct specif ic shRNA interference plasmid vectors targeted to DAL-1 gene mRNA. The constructed shRNA expression vectors of positive clones were validated by double enzyme digestion and DNA sequencing. Detect the change of DAL-1 mRNA with Q-PCR and DAL 1 protein expression with Western blot, to screen the clone which DAL-1 was steady inhibi ted. The change of cell proliferation activity was detected by MTT assay. To evaluate changes of migration and invasion in DAL-I silenced NCI H460 cell by Transwell migration and invasion model. Analyze the relationship between DAL-1 and NSCLC invasion. RESULTS： The se quences and plasmid were identificated by double-enzyme digestion and DNA sequencing. The mRNA expression level of DAL-1 of T4 group decreased （87.41±1.994）% respectively of that in NCI-H460/TN; Western blot analysis showed the DAL 1 protein decreased （82.73±2. 147）% of T4 group respectively of that in NCI-H460/TN as well. MTT assay showed that NCI H460/T cell proliferation was significant ly enhanced compared with that in NCI H460/TN （P=0. 000）. The capabilities of migration and invasion of NCI-H460/T cell significantly enhanced detected（P= 0. 000）. CONCLUSIONS= NCI-H460/T cell line which had successful and stable inhibition of DAL 1 expression is established successfully. Silencing of DAL-1 gene can enhance the proliferation, migration and invasion of NCI-H460 invitro, the DAL 1 expression can inhibit the proliferation, migration and invasion of NSCI.C.

关 键 词：癌 非小细胞肺 基因 DAL-1 RNA干扰 

分 类 号：R734.2[医药卫生—肿瘤]