| Nowadays breast cancer has ranked second after lung cancer and is harmful to people's health. The survival rates for breast cancer patients suffering 5 years and 10 years have increased to 70% and 50% respectisely, but about 40% of them still die of the cancer recurrence or metastasis. Recent research has found that the first requirement for the matastasis of breast cancer cells is the process of forming "invadopodia", which are actin-driven, finger-like membrane protrusions. The extracellular matrix proteins around it will be degraded by protease, which is released through the integration between cystic cancer and the cell membrane structure. And then it will allow the cancer cells to escape from the primary cancer. IBP (IRF-4 binding protein) is one of the proteins expressed in the immune system with important functions. IBP is involved in the formation of T cell-mediated immunity synapses after TCR signal stimulation. It is closely related with Th2 cell differentiation, affecting the function of Th17 cells by inhibiting IRF-4 transcription factor activity to regulate the production of IL-17 and IL-21. IBP played an important role in the maintenance of the immune environment stability. Loss of IBP in mice resulted in the development of systemic autoimmunity and developmental defects at the earliest stage of thymocyte differentiation. In addition, IBP plays a role of as a GEF, guanine nucleotide-exchange factor; IBP can activate some members of the Rho GTPase family. All these results indicate that IBP can take part in the reconstruction of the cytoskeleton, cell polarization, the signal transduction and many other important physiological processes. However, there is little research about the IBP expression and function presented within the body because of the lack of specific anti-IBP antibody.In this study, the IBP expressions in breast cancer and its possible clinical significances have been studied, and the function of IBP in breast cancer has been confirmed by in-vitro study. We studied the following three aspects: 1. The preparation and characterization of specific anti-IBP antibodiesFirst of all, the structure of IBP protein was analyzed by the bioinformatics method. The region with strong antigenicity was chosen for recombinant expression. The anti-IBP antibody was prepared by immunization BALB / C mouse, fusion and screening. In addition, the specific anti- IBP monoclonal antibody was determined by a variety of tests: ELISA, WB, IHC, ICC, and so on.2. The research of IBP expression in breast cancer and Its clinical significance(1) The study of IBP expression by WB and RT-PCR was based on breast cancer cell line of MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-453, MDA-MB-468, SK-BR-3.(2) The determination of IBP expression patterns in breast cancer was carried out by double immunofluorescence staining. The results showed that IBP expression patterns were primarily in the membrane and cytoplasm.(3) The construction of breast cancer tissue microarray was accomplished by collecting 107 cases of clinical breast cancer samples with different pathological classification.(4) The analysis of IBP expression in clinical samples was based on immunohistochemical staining, and the statistical analysis on IBP expression and other pathological indications to determine its clinical significance.3. The study of IBP function and mechanism in breast cancer(1) The construction of IBP over-expression model by plasmid transfection was based on MDA-MB-435 cells with IBP negative expression and IBP low-expression model made by RNAi method based on MDA-MB-231 cells with IBP positive expression.(2) The determination of the effects on cell growth was based on cell proliferation in each group, by the MTT assay.(3) The ability of cell invasion was based on analysis of stained-cells penetrating study by trans-well experiments; determining the effects of IBP on the ability of cell invasion.(4) The effects of IBP expression in breast cancer cells on the resistance to paclitaxel, which is commonly used in clinical treatment of breast cancer, was determined by MTT assay.(5) The relationship among IBP expression and cytoskeleton was based on the cytoskeleton staining, the relationship assay between IBP expression and the focal adhesion formation was based on immunofluorescence staining. (6) The determination of the relationship between IBP expression and Rac1b was based on double immunofluorescent staining.The results are as follow:1. We obtained 7 strains of hybridoma cells (A2, A9, C6, D5, F5, G3, and G4) excreting anti-IBP monoclonal antibody after ELISA and WB screening The mAb from five of them (A2, A9, C6, D5 and F5) could be applied to the IHC staining of IBP. Three antibodies (A9, C6 and D5) showed the strongest IHC staining.2. IBP expression was studied in five human breast cancer cell lines and was found positinely in MCF-7, MDA-MB-231 and SKBR3 cell lines at both the mRNA and protein levels. In addition, we confirmed that IBP located on the membrane of MCF-7 cells by A9 mAb and DiI double staining .3. Among the tissue samples analyzed here, 20 normal breast tissue samples, and 20 benign breast lesion samples were negative for IBP immunocytochemical staining with mAb A9. In contrast, the IBP protein was detected in the cytoplasm of many breast cancer cells, as indicated by the 57 IBP-positive samples among the 107 breast cancer tissue samples that were tested (53.3%). Based on the statistics analysis, we found that there was a significant positive correlation (P < 0.01) between the IBP expression level and the tumor grade as well as the presence of lymph node metastasis and also, there was a negative correlation between IBP and p53 protein levels (P< 0.05). Our results demonstrate that IBP is over-expressed in a considerable proportion of breast cancer cell lines and cancer tissues, and, therefore, IBP may be involved in breast cancer progression.4. Comparison of the growth rates of the tumor cells over a 9-day culture period revealed the MDA-MB-231 breast cancer cells with lower IBP expression grew more slowly (P<0.05) than cells that were transfected with the control siRNA construct. The IBP-over-expressing breast cancer cells grew much faster than their corresponding parental cells. To analyze the role of IBP in the invasiveness of breast cancer cells, a matrigel cell invasion assay system was used. This assay revealed that IBP-positive breast cancer cells exhibited greater penetration of extra matrix proteins when compared with IBP-negative tumor cells.5. With IBP over-expression the cell morphology of MDA-MB-435 changed significantly and invadopodia formation increased significantly. In contrast, with RNAi-induced IBP lower-expression, the cell invadopodia were greatly reduced. Further more, it has been found that IBP co-localized with microfilament. The co-localization was mainly situated on adhesion plaque, but the mainly situated was on larger peripheral focal adhesions with IBP low-expression.6. It was confirmed that there was synergistic effect between IBP and Rac1b based on double immunofluorescence staining.IBP was expressed not only in the immune system but also in breast cancer. IBP expression was closely related to the progression of breast cancer: IBP promotes the proliferation and invasion of breast cancer cells, the mechanisms of that may be that IBP would enhance the formation of cell invadopodia, participate in the construction of cell adhesion plaque to increase cell migration, and promote the generation of intracellular reactive oxygen species based on the synergistic effects with Rac1b, which allow the basal cells to achieve the transfer from the breakthrough. In our work, we tried to study the two primary requirements for the breast cancer's metastasis (invadopodia formation and basal lamina damage) by linking them to each other. It has been found that IBP was closely related to invadopodia formation and basal lamina damage, and it may be a bridge between both of them. The results of this study may improve the understanding of the occurrence and transfusion mechanisms of breast cancer and provide new targets and ideas for the clinical diagnosis and treatment early.
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