OBJECTIVE To study the expression levels of Twist and epithelialmesenchymal transitions in multidrug-resistant MCF-7/ADR breast cancer cells, and to study the relationship between multidrug resistance （MDR） and metastatic potential of the cells. METHODS RT-PCR, immunohistochemical and Western blotting methods were used to examine the changes of expression levels of the transcription factor Twist, E-cadherin and N-cadherin in the MCF-7 breast cancer cell line and its multidrug-resistant variant, MCF-7/ADR. RESULTS In MCF-7 cells, the expression of E-cadherin can be detected, but there is no expression of Twist or N-cadherin. In MCF-7/ADR cells, E-cadherin expression is lost, but the expression of two other genes was significantly positive. CONCLUSION Epithelial-mesenchymal transitions induced by Twist, may have a relationship with enhanced invasion and metastatic potential during the development of multidrug-resistant MCF-7/ADR breast cancer cells.
Fei Zhang Yurong Shi Lin Zhang Bin Zhang Xiyin Wei Yi Yang Rui Wang Ruifang Niu
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 PCRRESULTS 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.
Lin ZhangRui WangYurong ShiYi YangXiyin WeiZhi YaoRuifang Niu
OBJECTIVE The aim of this research was to clone and express the antigen of the previously prepared monoclonal antibody named M4G3. METHODS Western blots were used to screen a breast cancer cell line that overexpresses the M4G3-associated antigen. A λ, zap cDNA expression library of breast cancer cells was constructed and screened using M4G3 as a probe to clone the antigen. The positive clones were subcloned and identified by homologous comparison using BLAST. RESULTS The λ zap cDNA expression library had 1.0×10^6 independent clones. Fifteen positive clones were isolated following 3 rounds of immunoscreening and identified as being from Mycoplasma pulmonis. CONCLUSION The specific antigen that matched the monoclonal M4G3 antibody is an unknown protein of M. pulmonis. This work is helpful for the further study of the association of M. pulmonis infection with breast cancer.
Shuhua YangLin ZhongRuifang NiuDefa WangYurong ShiXiyin WeiYi Yang
