shRNA和siRNA敲降NET-1对A431细胞生物学行为影响的比较

Comparison of si RNA and sh RNA targeting NET-1 on the inhibition of the biological behavior of A431 cells

目的:比较小干扰RNA(small interference RNA,si RNA)和小发夹RNA(small hairpin RNA,sh RNA)敲降皮肤鳞癌细胞株(A431)中NET-1基因的表达,及其对癌细胞增殖、浸润的影响。方法:分别构建针对人NET-1基因的si RNA NET-1真核表达载体(p U6H1-GFP-si RNA NET-1,si RNA NET-1)和sh RNA真核细胞表达质粒(psilencer4.1-sh RNA NET-1,sh RNA NET-1),同时构建相应载体的随机序列(target-off)的对照质粒(sh RNA target-off和si RNA targetoff)。体外瞬时转染A431细胞,通过实时定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-q PCR)、Western Blot分别检测癌细胞内NET-1 m RNA和蛋白的表达,免疫荧光染色在镜下观察NET-1蛋白在细胞内的表达;3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide,MTT]法和流式细胞术检测细胞增殖;划痕试验和Transwell试验分别检测A431细胞的迁移和侵袭能力,比较si RNA和sh RNA对NET-1基因的抑制效果。结果:测序证实编码NET-1序列的si RNA和sh RNA已经分别插入载体p U6H1-GFP和psilencer4.1的启动子之间,测序结果符合设计要求。转染si RNA NET-1和sh RNA NET-1后,均能显著下调A431细胞NET-1 m RNA和蛋白的表达水平,显著抑制A431细胞的增殖、迁移和浸润,与对照组(target-off)比较差异均有统计学意义(均P<0.005)。在细胞生长曲线分析中显示转染sh RNA NET-1组在72 h后抑制细胞增殖的效果强于si RNA NET-1组。结论:NET-1基因的功能与A431细胞增殖、迁移、浸润有关。靶向NET-1的si RNA和sh RNA真核表达载体均能特异、有效地下调NET-1基因的功能。sh RNA介导的RNAi作用能更长期、稳定的抑制目的基因的表达,更适合于对目的基因功能进行长期研究。

Objective： To compare the effects of NET-1 gene inhibited by small interfering RNA（siRNA） and short hairpin RNA（shRNA） in human skin squamous cell carcinoma cell line A431. Methods： The specific targeting NET-1 eukaryotic expression vectors, pU6H 1 -GFP-siRNA NET- 1 （siRNA NET- 1） and psilencer4.1 -shRNANET- 1 （shRNA NET- 1）, were constructed respectively. In addition, the expression vectors with random sequences were constructed as controls（siRNA target-off and shRNA target-off）, which were transfected into A431 cells. The expression of NET-1 at the levels of mRNA and protein in A431 cells were detected by real time quantitative polymerase chain reaction（RT-qPCR） and Western Blot. Immunofluor-escence staining was used to observe NET-1 protein expression in cells. The cell proliferation was examined by 3-（4,5）-dimethyhhiahiazo（-z-yl）-3,5-di-phenytetrazoliumromide（MTY） assay and the cell cycle was analysed with flow cytometry; in vitro migration and invasion capability of A431 cells were evaluated by wound healing assay and transwell chamber experiments. Results： The eukaryotic expressing vectors of siRNA NET-1 and shRNA NET-1 were confirmed by sequencing and the results were consistent with the design requirements. After transfection in A431 cells, the expression of NET-1 at the levels of mRNA and protein were decresed; either siRNA or shRNA can obviously inhibit A431 cells proliferation, migration and invasion, respectively. Compared with the control group, the differences were statistically significant（all P〈0.005）, but there was significantly different between siRNA and shRNA group after transfection 48 hours in A431 cells proliferation level by MTT assay. Conclusion： Endogenous NET-1 gene is releated with A431 cell proliferation,migration and infiltration, siRNA and shRNA targeting NET-1 gene can specific and effective inhibit this gene function. In transiently transfected cells, siRNA and shRNA has the same effect, but the effects of cells proliferation by shRNA was longer than that by siRNA.