小RNA干扰VEGF-C基因表达对乳腺癌细胞增殖及凋亡的体外研究

VEGF-C siRNA induces apoptosis and inhibits growth of human breast cancer cell line MDA-MB-435

目的:利用质粒pSilencer3.0-H1构建人血管内皮生长因子-C(VEGF-C)基因经小RNA干扰(siRNA)的表达载体,体外研究其对人乳腺癌细胞MDA-MB-435的增殖及凋亡作用,为乳腺癌VEGF-C基因靶向RNA干扰治疗的可行性作初步探讨。方法:构建3组针对VEGF-C的pSilencer3.0-VEGF-C/siRNA表达载体和1组阴性对照序列。测序鉴定后,将脂质体介导转染人乳腺癌细胞株MDA-MB-435。用RT-PCR和Western Blot检测转染前后VEGF-C基因的表达。挑选干扰效率最强的一组表达载体。以MTT法和流式细胞仪检测经siRNA干扰的VEGF-C对人乳腺癌细胞MDA-MB-435的体外作用。结果:经测序鉴定,VEGF-C基因的siRNA表达载体构建成功,转染人乳腺癌细胞MDA-MB-435后,检测显示VEGF-C基因和蛋白水平表达量明显降低,抑制率最高达85.4%,细胞体外生长缓慢,72h后凋亡率达60%。结论:针对人VEGF-C基因的siRNA表达载体能抑制VEGF-C的基因表达,促进了人乳腺癌细胞MDA-MB-435的凋亡,可进一步发挥治疗作用。

Objective To construct recombinant small interfering RNA （siRNA） plasmid vector targeting VEGF-C, and observe its impact on the growth and apoptosis of human breast cancer cell line MDA-MB-435. Methods Three small fragments of siRNA and one negative control were sequenced and cloned into vector pSilencer3.0-H1. The recombinant plasmids were then transfected into breast cancer cell line MDA-MBA-435, by positive liposomal transfection assay. The transcription and translation level of VEGF-C expression were detected by RT-PCR and Western Blot. Meanwhile, the proliferation and apoptosis of transfected tumor cells were determined by MTI and flow-cytometry assay. Results The recombinant plasmids containing VEGF-C siRNA were successfully constructed. VEGF-C expression was significantly knocked-down after siRNA plasmid transfection. The maximum inhibitory rate of transfected breast cancer cells reached 85.4%, significantly higher that observed in the control group. The apoptosis rate was 60% 72hr after transfection. Conclusions VEGF-C gene silenced by siRNA can inhibit the proliferation and induce the apoptosis of human breast cancer cell line MDA-MBA-435. The process might serve as a potential approach for cancer gene therapy.