趋化因子受体4下调抑制乳腺癌转移的研究

Down-regulation of CXC chemokine receptor 4 Inhibits invasion of breast cancer cell line T47D

目的采用RNA干扰(RNAi)技术封闭乳腺癌细胞趋化因子受体4(CXCR4)基因,观察封闭效果及癌细胞的体外侵袭能力的变化及转移相关基因的变化。方法设计3段siRNA,分别构建pSilencer封闭载体,转染高表达CXCR4基因的乳腺癌细胞系T47D,通过流式细胞术及定量聚合酶链反应(PCR)的方法分别从蛋白水平和转录水平检测CXCR4表达率的改变;采用Matri- gel对转染后乳腺癌细胞体外侵袭能力的变化进行检测,应用定量PCR分析其他转移相关基因的变化。结果3组转染CXCR4-siRNA细胞定量PCR检测CXCR4抑制率分别为95.67%、85.94%、98.25%。流式细胞仪检测CXCR4表达率分别为4.95%、11.10%、5.53%,与转染Control-siRNA细胞比较差异有统计学意义(P<0.01)。3组封闭细胞的侵袭指数分别为0.037、0.290和0.188。封闭细胞E-钙黏蛋白(E-cad)表达有增加趋势,胰岛素样生长因子结合蛋白-5(IGFBP-5)表达有下降的趋势。结论CXCR4在乳腺癌转移中具有重要作用,mRNA具有强大的基因敲除能力,为肿瘤转移的基因治疗奠定实验基础。

Objective To design and construct small interfering RNA （siRNA） expression plasmids targeting CXCR4 gene, and detect the invasiveness of the CXCR4-siRNA T47D cell and the change of other metastasis-associated genes. Methods Three siRNAs were designed and ligated into the linearized pSilencer vector, respectively. The recombinant plasmids were transfected into T47D cells highly expressing CXCR4. The expression of CXCR4 mRNA and protein in the transfected cells was detected by flow cytometry and real-time quantitative PCR. Moreover,the cell invasion was evaluated using 24-well Matrigel invasion chambers. The changes of other metastasis-associated genes, such as E-cad, IGFBP-5, FN and MMP- 2,were identified by real-time PCR. Results The suppression rate of CXCR4 expression was 95.67%, 85.94% and 98.25% respectively in the CXCR4-siRNA cells by the analysis of Real-time PCR. There was statistically significant difference in the CXCR4 expression between control-siRNA cells and CXCR4- siRNA cells （P 〈0.01 ）. The invasion index of these CXCR4-siRNA cells was 0.037,0. 290 and 0. 188 respectively. After treatment of the cells with CXCR4-siRNA, the expression of E-cad showed an upward tendency and that of IGFBP-5 had a downward trend, while alteration in the expression of FN and MMP2 varied without a consistent effect. Conclusion CXCR4 played an important role in metastasis of human breast cancer, siRNA could powerfully trigger the CXCR4 gene silence in human breast cancer cell line T47D. This study was helpful for further study on the gene therapy for breast cancer metastasis.