| Purpose: As succeeding cloned ERs gene, peoples has been received a great achievements in the transcriptional mechanism study between the structure and introduction of ERa. Recently, it has been found another ER which called ER(3. However, the role of ERp in breast cancer is still unclear. In the present study, we transfected ERp gene into human breast cancer cells which lack of ERp gene expression to investigate its function. Also we will evaluate the relationship of angiogenesis factor bFGF with different ER subtypes and the prognostic value of ERp and bFGF in human breast cancer patients.Methods: By using lipofectAMINE method, we transfected ERp expression vector into MDA-MB-435 cells which lack the ERp gene. The transfected cells were subjected to purimycin (10^.g/ml), and selected anti-purimycin clones. The selected clones were conformed to have ERp gene and protein expression by RT-PCR and Western blot. Cell cycle was analyses by using flow cytometry, The cell cycle related proteins such as cyclin Dl, cyclin E and p21 were analyses by using Western Blot.In clinical study, 86 breast cancer patients were included in this study. ERa and PR immunoreactivities were measured in primary breast carcinoma excisional biopsies which were obtained from surgery. ERp and bFGF were detected by Western blots. Human fresh testis tissue was used as positive control for ERp. Bands for Western blots were quantitated using a Molecular Dynamics Laser Densitometer (Model PSD1) and an Image Quant Version 1 software program. For ERp, the band of testis was set to be 100, and bands of sample less than 50 was defined as low expression3and bands of sample more than 50 as high expression. For bFGF, the fetal liver was used as positive control of relative VEGF expression. As described above, normalized fetal liver bFGF expression as 100, and the bands of sample less than 100 was defined as low expression the bands of sample more than 100 as high expression. The difference of ERp protein expression levels between various clinical factors were evaluated using the chi-square test. The relationship of bFGF protein expression and different ER type was also studied using the chi-square test. The Kaplan-Meier method was utilized to estimate disease-free and overall survival times. The Wilcoxon (Gehan) test was used to assess the univariate effect of the expression of different ER subtypes and VEGF on relapse-free and overall survival times. The Cox's proportional hazard model was used to examine differences in overall survival and relapse-free survival after adjustment for other co-variates. The software used was SPSS 10.0 for windows.Results: Part I: (1), By using lipofectAMINE method, we transfected ERp expression vector into MDA-MB-435 cells which lack the ERp gene. Anti-purimycin clones were selected. The selected clones were conformed to have ERP gene and protein high expression by RT-PCR and Western blot. (2), We demonstrated that MDA-MB-435 cells which high expressed KRp protein have increased double time, and S phase comparing to it's control cells. (3), We also found that MDA-MB-435 cells which high expressed ERp protein significantly increased cyclin Dl and cyclin E expression. Part II: Of 86 patients, 59 cases (68. 5%) were ER(i protein low expressed, and 40 cases (46. 6%) were bFGF protein low expressed. When correlated the ERp protein levels with other clinical characters, no significance was found between ERp protein level and ERa expression and PR expression. When compared the bFGF levels with different ER subtypes, it was found4a significant difference among ER$^ ERa and bFGF. In ER0 protein high expression group and ERa low expression group, the bFGF protein was high expressed (p<0. 05). In univariate analysis ERa, ER(5 and bFGF levels had prognostic value for both relapse-free survival and overall survival (p<0.05). Also we analyzed the expression levels of ER(3 and bFGF affecting the survival of breast cancer patients. We found that the subgroup which both expressed high levels of ER(3 and b...
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