Down-Regulation of CXCR4 Expression by siRNA Inhibits Invasive Ability of Breast Cancer Cells

OBJECTIVE To investigate the efficiency of gene silencing by CXCR4- siRNAs (small interfering RNA), and to examine the invasive ability and the expression of other metastatic-associated genes in siRNA-treated breast cancer cells. METHODS Three siRNAs were designed and cloned into the pSilenc ? 3.1-H1 neo vector. The reconstructed plasmids were purified and trans- fected into the T47D breast cancer cell line, which highly expressed CXCR4. The amount of CXCR4 expression in the transfected cells was measured by flow cytometry and Real-time PCR. Cell invasive ability was evaluated us- ing 24-well Matrigel invasion chambers. In addition, the expression of other metastatic-associated genes, such as E-cad, IGFBP-5, FN and MMP-2, was assessed by Real-time PCR. RESULTS The suppression rates of CXCR4 mRNA expression reached 95.7%, 85.9% and 98.3%compared with control-siRNA cells in the 3 CXCR4- siRNA T47D cells respectively. FCM assays for CXCR4 protein expression showed a similar inhibitory effect. The invasion indexes of these CXCR4- siRNA cells were 0.037, 0.290 and 0.188 respectively compared with control- siRNA cells. 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 expression of FN and MMP2 varied without a con- sistant effect. CONCLUSION CXCR4 plays an important role in modulating migra- tion of human breast cancer cells. Small interfering RNA can significantly silence the CXCR4 gene in the human T47D breast cancer cell line. The results of this study strengthen the need for further research on novel gene therapy against breast cancer metastasis.

OBJECTIVE To investigate the efficiency of gene silencing by CXCR4- siRNAs （small interfering RNA）, and to examine the invasive ability and the expression of other metastatic-associated genes in siRNA-treated breast cancer cells. METHODS Three siRNAs were designed and cloned into the pSilenc ^TM 3.1-H1 neo vector. The reconstructed plasmids were purified and transfected into the T47D breast cancer cell line, which highly expressed CXCR4. The amount of CXCR4 expression in the transfected cells was measured by flow cytometry and Real-time PCR. Cell invasive ability was evaluated using 24-well Matrigel invasion chambers. In addition, the expression of other metastatic-associated genes, such as E-cad, IGFBP-5, FN and MMP-2, was assessed by Real-time PCR RESULTS The suppression rates of CXCR4 mRNA expression reached 95.7%, 85.9% and 98.3%compared with control-siRNA cells in the 3 CXCR4- siRNA T47D cells respectively. FCM assays for CXCR4 protein expression showed a similar inhibitory effect. The invasion indexes of these CXCR4- siRNA cells were 0.037, 0.290 and 0.188 respectively compared with control- siRNA cells. 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 expression of FN and MMP2 varied without a consistant effect. CONCLUSION CXCR4 plays an important role in modulating migra- tion of human breast cancer cells. Small interfering RNA can significantly silence the CXCR4 gene in the human T47D breast cancer cell line. The results of this study strengthen the need for further research on novel gene therapy against breast cancer metastasis.