VEGF-C短发夹RNA对人乳腺癌细胞的体内外抑制作用的实验研究

Short hairpin RNA-mediated inhibition of VEGF-C on human breast cancer cells in vitro and in vivo

目的:探讨靶向血管内皮生长因子-C(VEGF-C)的短发夹RNA(shRNA)质粒载体在体内外对乳腺癌细胞株MCF-7生物学活性的抑制作用。方法:倒置荧光显微镜及流式细胞术检测细胞的转染率,RT-PCR及免疫印迹法检测转染细胞内VEGF-C mRNA及蛋白的表达变化,MTT法检测细胞增殖抑制率,人工基底膜侵袭实验检测细胞侵袭能力。MCF-7裸鼠皮下尾静脉注射VEGF-C shRNA表达载体,观察其对乳腺癌生长的抑制作用。ELISA方法检测VEGF-C在肿瘤组织中的蛋白浓度,免疫组织化学分析肿瘤组织VEGFR-3阳性脉管密度。结果:VEGF-C shRNA能高效转染入MCF-7细胞中,细胞VEGF-C mRNA及蛋白表达水平显著下降,生长增殖受抑(F24 h=515.51,F48 h=859.97,F72 h=992.91,P均=0.000 1)。体外侵袭人工基底膜能力减弱(F=133.36,P=0.000 1)。全身系统性给予VEGF-C shRNA后,VEGF-C shRNA组肿瘤体积缩小(F=498.05,P=0.000 1);瘤组织VEGF-C浓度降低(t=30.75,P=0.001);免疫组化分析VEGF-C shRNA组肿瘤组织中VEGFR-3阳性脉管生成减少(F=265.49,P=0.000 1)。结论:VEGF-C在乳腺癌增殖、侵袭转移和VEGFR-3阳性脉管形成中发挥重要作用,通过RNA干扰技术实现VEGF-C沉默在乳腺癌的基因治疗中具有较好的应用前景。

OBJECTIVE： To investigate the anti-tumor effect of shot hairpin RNA（shRNA）-mediated inhibition of VEGF-C on human breast cancer in vitro and in vivo.METHODS：Transfection rate was observed by inverted fluorescence microscope and flow cytometry.The expression of VEGF-C was determined in transfected cells by RT-PCR and Western blot.The cell-growth inhibition rate and the invasive capacity were evaluated by the MTT method and reconstituted basement membrane invasion assay.VEGF-C shRNA vector was injected from the tail vain of the tumor model mice after tumor cell innoculation.The concentration of VEGF-C in tumor homogenates was detected by ELISA.VEGFR-3 positive vessels in tumor tissues were measured by immunohistochemical analyses.RESULTS： VEGF-C shRNA specifically and effectively downregulated VEGF-C mRNA and protein expression in vitro,and it also effectively inhibited proliferation（F24 h=515.51,F48 h=859.97,F72 h=992.91,P=0.000 1） and invasive capacity（F=133.36,P=0.000 1） of MCF-7 cells.The treatment with VEGF-C shRNA in vivo suppressed tumor growth significantly,F=498.05,P=0.000 1;VEGF-C ELISA of tumor homogenates confirmed RNA interference（t=30.75,P=0.001）;immunohistochemical analyses demonstrated significant reduction of VEGFR-3 positive vascular formation in tumor tissues（F=265.49,P=0.000 1）.CONCLUSIONS： VEGF-C plays an important role in tumor growth,invasive capacity and lymphangiogenesis of breast cancer.RNA interfering target VEGF-C may serve as potential therapeutic interventions in human breast cancer.