VEGF-C 短发夹RNA 抑制乳腺癌细胞增殖及侵袭能力的实验研究

doi:10.3877/cma.j.issn.1674-0807.2008.01.010

中华乳腺病杂志(电子版) ›› 2008, Vol. 02 ›› Issue (01) : 32 -35. doi: 10.3877/cma.j.issn.1674-0807.2008.01.010

实验研究

VEGF-C 短发夹RNA 抑制乳腺癌细胞增殖及侵袭能力的实验研究

古立诚¹, 肖焕擎²^,^†(

), 徐波², 朱光辉², 李书华³

1.510620 广州,广州市第十二人民医院普外科
2.510120 广州,广州医学院附属广州市第一人民医院普外科
3.510182 广州,广州医学院病理教研室

收稿日期:2007-11-01 出版日期:2008-02-01
通信作者: 肖焕擎
基金资助:
广东省社会发展领域科技计划项目(2006B35530001)

Down-regulation of VEGF-C using short hairpin RNA inhibits proliferation and invasion of human breast cancer cells

Li-cheng GU¹, Huan-Qing XIAO^†^,¹(), Guang-hui ZHU¹, Shu-hua LI¹

1.Department of General Surgery,Guangzhou Municipal Twelfth People's Hospital,Guangzhou 510620,China

Received:2007-11-01 Published:2008-02-01
Corresponding author: Huan-Qing XIAO

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目的

探讨应用靶向VEGF-C 的短发夹RNA(shRNA)质粒载体在体外抑制乳腺癌细胞株MCF-7 生物学活性的影响。

方法

制备携带有绿色荧光蛋白(GFP)报告基因的VEGF-C-shRNA质粒载体,脂质体转染方法转入乳腺癌细胞株MCF-7,通过倒置荧光显微镜及流式细胞术检测细胞的转染效率,RT-PCR 及Western blot 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,重组基底膜侵袭实验检测细胞侵袭能力。

结果

高效转染入VEGF-C-shRNA 的MCF-7 细胞VEGF-C mRNA 及蛋白表达水平显著下降;VEGF-C ShRNA 对MCF-7 细胞具有明显的增殖抑制作用,其抑增殖效应呈时间依赖性;VEGF-C-shRNA 可抑制乳腺癌细胞MCF-7 体外侵袭重组基底膜的能力,与阴性shRNA 转染组、空载体组及空白组比较,差异有统计学意义(P＜0.05);

结论

VEGF-C在乳腺癌增殖、侵袭转移中发挥重要作用,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可行性。

关键词: 血管内皮生长因子-C, 乳腺癌, 短发夹RNA, RT-PCR, Western blot

Objective

To investigate the anti-tumor effect of short hairpin RNA (shRNA)-mediated inhibition of VEGF-C (vascular endothelial growth factor C) on human breast cancer in vitro.

Methods

Breast cancer cell line MCF-7 was stably transfected with a VEGF-C short hairpin RNA(shRNA) plasmid vector labeled with green fluorescent protein. The transfected cells were visualized by fluorescent microscope and assayed by flow cytometry. The expression of VEGF-C in transfected cells was determined by RT-PCR and Western blot. The cell growth inhibition rate and the invasive capacity were evaluated by MTT method and reconstituted basement membrane invasion assay.

Results

VEGF-C shRNA specificly and effectively downregulated VEGF-C mRNA and protein expression in vitro. And it also effectively inhibited proliferation and invasive capacity of MCF-7 cells when compared to vehicle,vector and control shRNA.

Conclusions

These data in our study demonstrated that VEGF-C plays an important role in tumor growth,invasive capacity of breast cancer.RNA interference targeting VEGF-C may serve as a potential therapeutic intervention for human breast cancer.

Key words: Vascular endothelial growth factor-C, Breast carcinoma, Short hairpin RNA, RTPCR, Western blot

表1 转染shRNA 后不同时点MCF-7细胞的增殖抑制率 (%)

分组	24h	48h	72h
VEGF-C-shRNA组	18.72±1.34^a	25.24±2.36^a	42.48±3.32^a
阴性shRNA组	6.26±0.16	6.42±0.29	6.79±0.21
空载体组	5.24±0.32	5.39±0.43	5.72±0.16
空白组	5.37±0.43	5.43±0.18	5.65±0.25

表1 转染shRNA 后不同时点MCF-7细胞的增殖抑制率 (%)

分组	24h	48h	72h
VEGF-C-shRNA组	18.72±1.34^a	25.24±2.36^a	42.48±3.32^a
阴性shRNA组	6.26±0.16	6.42±0.29	6.79±0.21
空载体组	5.24±0.32	5.39±0.43	5.72±0.16
空白组	5.37±0.43	5.43±0.18	5.65±0.25

表2 转染shRNA 对乳腺癌MCF-7 细胞侵袭重组基底膜能力的影响

分组	侵袭细胞数	抑制率/(%)
VEGF-C-shRNA组^a	56±3.2	42.9
阴性shRNA组	97±3.2	-
空载体组	98±7.4	-
空白组	98±2.0	-

表2 转染shRNA 对乳腺癌MCF-7 细胞侵袭重组基底膜能力的影响

分组	侵袭细胞数	抑制率/(%)
VEGF-C-shRNA组^a	56±3.2	42.9
阴性shRNA组	97±3.2	-
空载体组	98±7.4	-
空白组	98±2.0	-

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