The Preliminary Study On Effect Of Down-regulation Of CXCR4 On Breast Cancer Metastasis

Background & ObjectiveMorbidity and mortality in cancer are mainly determined to organ-specific metastasis and the failure of chemotherapeutic drugs to selectively kill cancer cells at the sites of metastasis. Metastasis is a non-random process, and each cancer type has its own preferred sites of metastasis. For instance, breast cancer cells preferentially metastasize to the regional lymph nodes, lungs, liver, and bone. Prostate cancers usually metastasize to bone. While there has been considerable progress in identifying genes that promote the metastasis of cancer cells, little is known about the genes that enable cancer cells to seed, survive, and proliferate at sites of metastasis. Muller and colleagues showed that metastatic breast cancer cells overexpress the chemokine receptor CXCR4. Additionally sites to which breast cancer cells metastasize express abundant amounts of stromal derived factor-1 (SDF-1, recently renamed CXCL12), the ligand for CXCR4. Moreover functional blockade of CXCR4 by antibodies significantly inhibited lymph node and lung metastasis in xenograft models of breast cancer. These results suggest that SDF-1 serves as a homing factor for cancer cells and that the signaling pathways activated upon interaction of SDF-1 with its exclusive receptor CXCR4 play an important role in the survival and proliferation of cancer cells once they are localized in a specific organ. RNA interference technology, silencing targeted genes specifically and high-efficiency in mammalian cells, has become a powerful tool for studying gene function. In my study, the human breast cancer cell line T47D with its overexpressing CXCR4 was selected and transfected with stable siRNA expressing system for the first time in cancer research. Then, we tested the ability of invasion of the transfected T47D in vitro. This study is aimed to exploita new way to anti-metastasis. MethodsIn the first part, we used a human breast carcinoma cell line (T47D cell), which could highly express the receptor of CXCR4. During the beginning of siRNA design, we selected four target sites from the full sequence of CXCR4 gene according to a scanning program on a web (http://iura.wi.mit.edu/siRNAext/). Then the 3' adjacent 19 nucleotides as potential siRNA target sites were entered into the web-based insert design tool at the following address: http://www.ambion.com/techlib/misc/psilencer converter.html. After the program of designing was completed, there were eight complementary hairpin siRNA template oligonucleotides. They must be synthesized, annealed, and ligated into the linearized pSilencer vector for each siRNA target site. Four plasmids, the ligation products, such as pSliencer 3.1-HI neo/siRNAl, pSliencer 3.1-HI neo/siRNA2, pSliencer 3.1-HI neo/siRNA3 and pSliencer 3.1-HI neo/siRNA4, were forming. These plasmids were transformed into E.coli in order to clone and amplify them. Then, we picked clones, isolated plasmid DNA, digested plasmids with BamH I and Hind III, and sequenced the plasmids to confirm the presence of the siRNA insert. Consequently, we purified the conformed plasmids, transfected them into T47D cells, and selected G418-resistant cells. Finally, the amount of CXCR4 expression in the transfected cells was measured by flow cytometry and real-time quantitative PCR.In the second part, we observed the proliferation ability of untransfected and transfected T47D cells through drawing cell growth curve by MTT colorimetric assay and clone-forming efficiency assay. Moreover, an assay of early-stage apoptosis was performed by flow cytometry. In addition, cell invasion wasevaluated using 24-well matrigel invasion chambers. ResultThe result of the first part:1 We were succeeded to design four pSilencer siRNA expression vectors, and the result of analysis of digested products and sequencing showed that all the insert sequences were right.2 During the process of selecting G418-resistent cells, only T47D cells transfected with pSilencer/siRNA2 died completely, and several clones were choosed from others (including T47D/siRNAl, T47D/siRNA3 and T47D/siRNA4).3 The rates of CXCR4 expression are as follows: 69.00% of parental T47D cells, 4.95% of T47D/siRNAl, 11.10% of T47D/siRNA3 and 5.53% of T47D/siRNA4. There was statistically significant deference in CXCR4 expression between parental T47D cells and tranfected cells (P<0.01), while no significant deference was found among three transfected T47D cells (P>0.05).4 The suppression rates of CXCR4 expression is 95.67%, 85.94%and 98.25% in the transfected cells by the analysis of RT-PCR.The result of the second part:1 The result of analysis of cell growth curve indicated that the ability of growth and proliferation of parental T47D cells was higher than that of transfected cells. In addition, the result of analysis of clone-forming assay showed that no significant deference in clone-forming efficiency between parental T47D cells and transfected cells (P>0.05).2 No early-stage apoptosis was observed in transfected and untransfected T47D cells.3 The invasion index of transfected cells are 0.037, 0.290 and 0.188 in matrigel...