摘要
目的利用RNA干扰技术下调趋化性细胞因子受体CXCR4基因的表达,探讨其沉默对乳腺癌细胞体外侵袭及肺转移潜能的影响。方法设计合成CXCR4的特异性短发卡状RNA(shRNA),将其插入至pSilencer载体中,并将重组后的pSilencer质粒载体经脂质体包裹转染乳腺癌MDA—MB-231细胞株,抗性筛选稳定抑制CXCR4表达的永久细胞克隆。应用逆转录聚合酶链反应(RT—PCR)和Western blot法,检测shRNA对细胞内CXCR4基因表达的影响。利用Boyden小室模型检测细胞体外侵袭能力。二苯基溴化四氮唑蓝(M1TF)法检测细胞的增殖状况。通过裸鼠尾静脉瘤细胞注射方法,构建肺转移模型,检测CXCR4基因沉默对乳腺癌细胞肺转移能力的影响。结果成功构建和筛选出CXCR4特异性的shRNA质粒载体,稳定转染CXCR4-shRNA的乳腺癌细胞的CXCR4mRNA和蛋白的表达较空白对照组明显下调(29.5%±3.8%比69.7%±2.6%.15.4%±1.1%比39.0%±2.4%;均P〈0.01),体外侵袭能力减弱,增殖速度减慢,肺转移能力下降。结论以CXCR4为靶向的shRNA能够有效下调CXCR4基因的表达,降低人乳腺癌细胞体外侵袭、增殖以及肺转移的能力。CXCR4是乳腺癌侵袭和转移过程中的重要调控因子。
Objective To construct a CXCR4 specific recombinant plasmid vector and study its inhibiting effect on invasion capacity in vitro of human breast cancer MDA-MB-231 cell line and its metastatic potential to the lung in nude mice. Methods A CXCR4 specific recombinant plasmid vector was constructed and transfected into the cultured MDA-MB-231 cell line with lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of CXCR4, respectively. Invasion capability in vitro of the cells was evaluated by Boyden chamber. The cell proliferation capacity was detected by MTT method. The nude mouse model of lung metastasis was established by injection of MDA-MB-231 cells into the tail vein. The animals were sacrificed at 6 weeks after the tumor cells injection. Whole lung tissues were harvested, embedded in paraffin, sectioned serially, and the HE-stained paraffin sections were examined pathologically to evaluate the presence and number of metastatic tumors. Results The CXCR4 mRNA expression rate was 29.5 % ± 3.8% in the CXCR4-shRNA group, significantly lower than that of the control group (69.7% ± 2.6%, P 〈 0.01 ) and mock-control group ( 67.8%± 3.5 %, P 〈 0.01 ). The CXCR4 protein expression rate was 15.4% ± 1.1% in the CXCR4-shRNA group, significantly lower than that of the control group (39.0% ± 2.4% , P 〈 0.01 ) and mock-control group ( 35.9% ± 3.9%, P 〈 0.01 ). Silencing of CXCR4 by shRNA lead to a significant decrease in breast cancer cell invasion and proliferation capacity in vitro. Furthermore, tumor cells with CXCR4 shRNA permanent transfcetion had a much lower lung metastatic potential in nude mice than control cells and mock control cells in vivo. Conclusion CXCR4 shRNA can inhibit the expression of CXCR4 and decrease the invasion and lung metastatic potential of human breast cancer cells.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2008年第5期325-329,共5页
Chinese Journal of Oncology
基金
国家自然科学基金资助项目(30570961)