shRNA沉默EIF4E基因对乳腺癌MDA-MB-231细胞VEGF-C表达的影响

孙阳阳; 曹友德; 刘浩; 叶维霞

doi:10.3969/j.issn.1000-8179.2011.04.006

摘要: 目的：探讨shRNA沉默EIF4E基因对乳腺癌MDA-MB-231细胞EIF4E及VEGF-C基因表达的影响。方法：制备针对EIF4E的三种小干扰RNA （small interfering RNA， siRNA），分别是siRNA1、 siRNA2、 siRNA3，筛选沉默最有效的siRNA，据此设计短发夹RNA （short hairpin RNA， shRNA）寡核苷酸链，与真核表达载体pGPU6/GFP/Neo克隆连接，构建pGPU6/GFP/Neo-EIF4E重组质粒，经酶切鉴定后利用脂质体LipofectamineTM2000将鉴定正确的重组质粒转染人乳腺癌MDA-MB-231细胞，荧光显微镜观察转染效率， RT-PCR、 Western blot、免疫细胞化学法检测EIF4E及VEGF-C mRNA和蛋白表达情况。结果： RT-PCR结果显示siR?NA1、 siRNA2、 siRNA3对乳腺癌MDA-MB-231细胞EIF4E基因表达均有抑制作用，与空白对照组、阴性对照组、脂质体组比较差异显著（P<0.05），尤以siRNA3抑制率最高达71.2%。利用针对siRNA3合成的shRNA与质粒载体pGPU6/GFP/Neo克隆连接构建的重组质粒pGPU6/GFP/Neo-EIF4E，酶切鉴定表明构建成功，并稳定转染MDA-MB-231细胞，平均转染效率为80%。RT-PCR、Western blot结果显示，稳定转染重组质粒后MDA-MB-231细胞EIF4E及VEGF-C mRNA和蛋白表达均明显下降（P<0.05），抑制率分别为79.00%、 67.90% （EIF4E）和77.01%、 62.94% （VEGF-C）。免疫细胞化学染色法显示重组质粒组EIF4E及VEGF-C蛋白表达均受到明显抑制（P<0.05）。结论：靶向EIF4E的shRNA不仅能有效沉默乳腺癌细胞MDA-MB-231中EIF4E的表达，且能抑制VEGF-C的表达，提示EIF4E可能参与诱导乳腺癌细胞VEGF-C的表达，而EIF4E-shRNA可能成为抑制乳腺癌淋巴管生成的一种新的有效手段。MDA-MB-231 RNA EIF4E pGPU6/GFP/Neo

Abstract: Inhibitory Effect of Down-regulation of EIF4E by ShRNA on the Expression of VEGF-C inBreast Cancer Cell Line MDA-MB-231Yangyang SUN, Youde CAO, Hao LIU,Weixia YECorresponding author: Youde CAO, E-mail: cydcyj@163.comDepartment of Pathology, Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, ChinaAbstract Objective: To study the effect of down-regulation of EIF4E by ShRNA on the expression of VEGF-C of breast cancerMDA-MB-231 cells. Methods: Three pairs of small interfering RNA (siRNA1, siRNA2, and siRNA3) were designed and prepared. Af-ter screening for the most efficient siRNA, its precursor short hairpin RNA (shRNA) was designed and cloned into plasmid pGPU6/GFP/Neo eukaryotic expression vector. The recombinant expression plasmid pGPU6/GFP/Neo-EIF4E was constructed and was identi-fied by enzyme restriction. The identified pGPU6/GFP/Neo-EIF4E plasmid was transfected into breast cancer MDA-MB-231 cells withLipofectamineTM2000. Fluorescence microscopy was used to observe the efficiency of tranfection. RT-PCR,Western-blot, immunohis-tochemistry and immunnofluorescence were performed to detect the expression of EIF4E and VEGF-C. Results: siRNA1, siRNA2 andsiRNA3 inhibited the expression of EIF4E gene in MDA-MB-231 cells, significantly different from the blank control group, negativecontrol group and liposome group (P<0.05). siRNA3 showed the highest inhibitory rate ( 71.2% ). The recombinant plasmid pGPU6/GFP/Neo-EIF4E was successfully constructed using shRNA and pGPU6/GFP/Neo, and it was stably transfected into MDA-MB-231cells, with the efficiency of transfection of 80%. RT-PCR and Western blot assay showed that stable transfection of the recombinant ex-pression plasmid resulted in reduction of EIF4E and VEGF-C mRNA and protein expression. The inhibitory rates weere 79% and67.9% ( EIF4E ) and 77.01% and 62.94% ( VEGF-C ), respectively, significantly different from the blank control group and empty vec-toe group. Immunohistochemistry and immunnofluorescence assay showed that the protein expression of EIF4E and VEGF-C was sig-nificantly inhibited ( P < 0.05 ) after transfection. Conclusion: shRNA -EIF4E not only silence the expression of EIF4E in breast cancerMDA-MB-321cells, but restrain VEGF-C expression, implying that EIF4E possibly participates the induction of VEGF-C expression,and suggesting that the technology may be an effective method to prevent lymphangiogenesis in breast cancer.Keywords MDA-MB-231 cells; Small interfering RNA ; EIF4E; pGPU6/GFP/Neo