摘要
目的探讨小分子干扰核酸（siRNA）对皮肤鳞状细胞癌（简称皮肤鳞癌）细胞株（A431）中NET-1基因的抑制作用，及其基因对癌细胞增殖、浸润的影响。方法构建针对人NET-1基因的siRNA NET-1真核表达载体（pU6H1-GFP—siRNANET-1），转染A431细胞后通过半定量RT—PCR检测细胞中NET-1 mRNA水平以筛选较有效的siRNA NET-1。同时设置针对NET-1的正、反义真核表达载体和随机序列对照组siRNA表达载体，体外瞬时转染A431细胞，RT—PCR、Western blot分别检测癌细胞内NET-1mRNA和蛋白的表达，经免疫荧光染色在激光共聚焦显微镜下观察NET-1蛋白在细胞内的表达；四甲基偶氮唑盐（MTT）法和流式细胞仪分别检测A431细胞增殖与占不同细胞周期中细胞增殖指数（PI）；划痕试验和Transwell迁移试验分别检测A431细胞的迁移和侵袭能力。结果测序证实编码NET-1序列的siRNA已经插入载体pU6H1-GFP中u6和H1两个启动子之间，其他载体测序结果符合设计要求。pU6H1-GFP载体转染A431细胞后其转染率达到80％。转染siRNA NET-1和反义NET．1后分别与未转染组比较，A431细胞中NET-1mRNA分别减少72％和62％（t值分别为-36．01，-17．65；均P〈0．05）和蛋白表达水平分别减少61％和69％（t值分别为-21．13，-33．14；均P〈0．05）；在转染48h后能显著抑制A431细胞的增殖、迁移和浸润（均P〈0．05）。在转染对照组siRNA后并未见到明显的抑制效果。转染正义NET-1后，能分别增加细胞内52％NET-1mRNA和49％蛋白表达水平（t值分别为12．49，13．98，均P〈0．01）。结论靶向NET-1的siRNA真核表达载体能特异、有效的下调NET-1基因蛋白的表达，并抑制A431细胞增殖、迁移和浸润。进而证明内源性NET-1基因的功能与A431细胞增殖、迁移、浸润有关。靶向NET-1的siRNA显示基因抑制效率比反义核酸技术更好。
Objective To investigate the effect of NET-1 siRNA on NET-1 expression and on the proliferation and infiltration of skin squamous cell carcinoma cell line A431. Methods Four recombinant vectors of pU6H1-GFP-siRNAs NET-1 were transfected into A431 cells. The levels of NET-1 mRNA expression were measured by semi-quantitative RT-PCR for selecting the most effective one in the four kind vectors of pU6H1-GFP-siRNA-NET-1. The controls consisted of pU6H1-GFP-siRNA-target off with random double-stranded RNA, pcDNA3.1 sense and other controls including the empty vectors （pU6H1-GFP and pcDNA3.1 ）, NET-specific siRNA alone, lipofection reagent alone, target off siRNA alone, or untreated cells. After transfection, levels of NET-1 mRNA and protein expression were detected using semi-quantitive RT-PCR and Western blotting, respectively. The intracellular location of NET-1 protein was documented by immunofluorescence staining followed by laser scanning confocal microscopy. Proliferation rates of A431 cells were determined by MTT assay and flow cytometry. Abilities of migration and infiltration of A431 cells were determined by wound healing effect and transwell migration assay, respectively. Results siRNAs coding NET- 1 sequences（19-23nt）were confirmed between HI and U6 promoters of pU6H1-GFP vector by sequencing. The transfection efficiency of pU6HI-GFP in A431 cells was about 80% as the percentage of GFP expression cells in total cells. Transfection with either pU6H1-GFP-siRNA NET-1 or pcDNA3. 1 antiseuse NET-1 gave rise to an obvious reduction in the expression of NET-1 mRNA and protein in A431 cells. The abilities of proliferation, migration and infiltration of A431 cells with either pU6H1-GFP-siRNA NET-1 or peDNA3.1 were significantly reduced 48 hours after transfection antisense NET-I, compared with the vector controls including pU6H1-GFP or pcDNA3.1 （ P 〈 0. 01, respectively） . pU6H1-GFP-siRNA NET-1 showed a stronger inhibition effect on proliferation, migration and infiltration of A431 cells over pcDNA3. 1 antisense NET-1 （P 〈 0.05）. Transfection with pcDNA3.1 sense NET-1 resulted in an obvious increase in the expression level of NET-1 mRNA and protein, but without inhibition effect to proliferation, migration and infiltration of A431 cells. No obviously inhibition effects were observed when transfected with pU6H1-GFP- siRNA-target or the controls. Conclusions RNAi targeting NET-I gene effectively down-regulates the expression of NET-1 in A431 cells, leading to an inhibition of proliferation, migration and infiltration. RNAi technique appears to be superior to antisense oligonucleotide technique in the suppression of gene expression.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2009年第10期691-696,共6页
Chinese Journal of Pathology
关键词
RNA
小分子干扰
寡核糖核苷酸类
反义
细胞系
皮肤肿瘤
癌
鳞状细胞
RNA, small interfering
Oligoribonucleotides, antisense
Cell line
Skin neoplasms
Carcinoma, squamous cell
