| Objective:Breast cancer is the most common malignant tumor of female in the world, which seriously threatening those health and lives. In recent years, the incidence of breast cancer keeps on rising in China and the age of onset is younger. Breast cancer is the endocrine dependent tumor. Estrogen can stimulate the occurrence and development of breast cancer, and to increase the probability of breast cancer recurrence and metastasis. Compared with chemotherapy drugs, endocrine therapy has become one of the most important treatment for breast cancer due to its little side effect, convenient taking, lasting effect and has small impact on quality of patients’ life. Tamoxifen(TAM) is a selective estrogen receptor modulators, which used to therapy the premenopausal breast cancer patients with estrogen receptor(ER) positive. At present, it was proved that about 40%~50% ER positive breast cancer patients became resistant of estrogen therapy. But the drug resistance mechanism of TAM is not clear, The loss and mutation of ER can’t fully explain the resistance of TAM.Based on our previous study, we find that as a kind of anti estrogen drugs, Tamoxifen can regulation the expression of Cerb B-2 gene, then effect the growth process of breast cancer cells. There are two genes: PAX-2(Paired box gene) and AIB-1(Amplified in breast cancer 1, also known as SRC-3) plays a very important role in the mediated. Study found that PAX-2 and AIB-1 can competitive binding ER to regulation the transcription of HER-2, which determines the effectiveness of tamoxifen in breast cancer endocrine therapy. In view of this, we carried out a systematic experimental study to explain the role of PAX2 and AIB1 expression in human breast cancer cell and the effect on drug resistant of tamoxifen.Methods:1 Establishment of a breast cell line MCF-7 stably transfected by over-expression PAX-2,AIB-1.1.1 Stably transfect PAX-2,AIB-1 gene recombinant plasmid into human breast carcinoma cell line MCF-7 by lipofectin medium, after screened with puromycin 4-5 weeks, we establish there new breast cancer cell lines stable express PAX-2 gene,AIB-1 gene(Named for MCF-7- PAX-2,MCF-7- AIB-1). Using the MCF-7 cell lines which stably transfected negative plasmid as controls(named for MCF-7-empty). The efficiency of stably transfection through by detecting the express of the green fluorescent protein GFP.1.2 The groups of the experiment: no treatment group(MCF-7 cell group); the control group(MCF-7-empty cell group); interference group 1(MCF-7-AIB1 cell group); interference group 2(MCF-7-PAX2 cell group).Use RT-PCR, Western blot methods to detect the expression of PAX2 m RNA,AIB-1 m RNA and protein of MCF-7- PAX2,MCF-7- AIB1 and MCF-7-empty to verifiy the stable cell line obtain.2 Study of the Proliferation and invasion of Effects and Drug resistance of the Stably Transfection Cell2.1 Establish the stable transfectants in nude mice model and measurement the tumor volumeMake the there transfected cells into single cell suspension,1.5×107 cells were injected subcutaneously in nude mice each right back area. Observe the growth of tumor after inoculation, calculated the tumor volume by the formula: TV=1/2×LW2. Given tamoxifen gavage until 7 weeks after inoculation, then observed the tumor growth after treatment.2.2 Preparation nude mice with translating tumorThe subcutaneous tumors were harvested at 12 weeks Post-injection. Remove the subcutaneous tumor specimens. HE staining to identify the cellular origin of the neoplasm. Meanwhile, the total RNA and protein were extracted from tumor tissue to detect PAX2,AIB1,HER2 and Ki-67 expression in the tumor. Using immunohistochemical to detected the specific expressionof PAX2, AIB1, HER-2, Ki-67.3 Statistical analysisFor each protocol, at least three independent experiments were performed. Results were expressed as the mean ± standard error of the mean(SEM). Statistical calculations were performed with the one way analysis of variance(ANOVA) and Nonparametric using SPSS 13.0 software. P<0.05 was considered statistically significant.Results:1 Purification of EX-Z4450-M90(PAX-2), EX-Z5301-M90(AIB-1), EX-NEG-M90(empty) plasmid was identified by agarose gel electrophoresis, and obtain the strips the same size of purpose. Determination by UV spectrophotometer method:EX-Z4450-M90(PAX-2) concentration 684μg/ml, OD260/OD280 value is 1.956; EX-Z5301-M90(AIB-1) concentration 598μg/ml, OD260/OD280 value is 1.942. The negative control plasmid concentration 962 μg/ml, OD260/OD280 value is 1.949, the ratio is in the reasonable range. Prove extraction of plasmid is successed, and plasmid is high purity to transfect.2 Through detecting the fluorescent protein GFP, the stably transfected efficiency of MCF-7-PAX2, MCF-7-AIB1 and MCF-7-empty is 80%.3 Result of RT-PCR show that: Compared to the no treatment group, PAX-2m RNA expression level of MCF-7- PAX-2 cell are significantly induced to 321.08 times(P<0.05); AIB-1 m RNA expression level of MCF-7-AIB-1 cell are significantly induced to 599.67 times(P<0.05); but no treatment group compared to the control group with no significant difference(P > 0.05).4 Result of Western blot show that: Compared to the no treatment group, PAX-2 protein expression level of MCF-7-PAX2 cell are significantly induced 67.7%(P<0.05); AIB-1 protein expression level of MCF-7- AIB1 cell are significantly induced 144.56%(P<0.05), but no treatment group compared to the control group with no significant difference(P>0.05).5 Result of tumor volume show that:①3 weeks after inoculation all the four groups of mice were had tumors.②5 weeks after inoculation, the tumor volume was different. Compared to the no treatment group, the tumor volume of MCF-7-PAX2 was significantly dropped 7.36%(P<0.05), the tumor volume of MCF-7-AIB1 was significantly induced 13.09%(P<0.05), but no treatment group compared to the control group with no significant difference(P>0.05).③7 weeks after inoculation, compared to the no treatment group, the tumor volume of MCF-7-PAX2 was significantly dropped 15.69%(P<0.05), the tumor volume of MCF-7-AIB1 was significantly induced 53.57%(P<0.05), but no treatment group compared to the control group with no significant difference(P>0.05).④5 weeks after TAM gavage: compared to the no treatment group, the growth rate of MCF-7-PAX2 was significantly dropped 27.5%(P<0.05), the growth rate of MCF-7-AIB1 was significantly induced 125.75%(P<0.05), but no treatment group compared to the control group with no significant difference(P>0.05).6 Result of HE staining and immunohistochemical show that:①Tumors were 4-μm-thick sections from formalin-fixed, paraffin-embedded tissue blocks were stained with H&E staining and immunohistochemical examination, HE staining of subcutaneous tumors treatment group and positive control cells formed no differences were seen high mitotic tumor cells arranged in dense shape nearly oval or polygonal.② Immunohistochemistry showed that all the four group PAX-2,AIB-1,HER-2 and proliferation-related factors of ki-67 were positive expression. Compared to the no treatment group, the tumor of MCF-7-PAX2 group’s PAX-2 strong positive expression rate is higher(P<0.05), HER2 strong positive expression rate is lower(P<0.05), there was no significant difference in AIB-1, Ki-67(P>0.05); the tumor of MCF-7- AIB-1 group’s AIB-1 strong positive expression rate is higher(P<0.05), there was no significant difference in HER2,AIB-1, Ki-67(P>0.05); but no treatment group compared to the control group with no significant difference(P>0.05).7 Result of tumor tissue RT-PCR show that: Compared to the no treatment group, the tumor of MCF-7-PAX-2 group’s PAX-2m RNA expression level is significantly induced 13315.24%(P<0.05),AIB-1m RNA and HER-2m RNA expression level are respectively decreased 67.16%, 54.63%(P<0.05); the tumor of MCF-7-AIB1 group’s AIB-1m RNA and HER-2m RNA expression level are respectively induced 5801.77%, 538.49%(P<0.05); PAX-2m RNA expression level is significantly decreased 28.90%(P<0.05); but no treatment group compared to the control group with no significant difference(P>0.05).8 Result of tumor tissue Western blot show that: Compared to the no treatment group, the tumor of MCF-7-PAX2 group’s PAX-2 protein expression level are significantly induced 91.06%(P<0.05), AIB-1 and HER2 protein expression level are respectively decreased 33.86%,38.65%(P<0.05); ki-67 protein expression level have no difference(P>0.05); the tumor of MCF-7-AIB1 group’s AIB-1 and HER2 protein expression level are respectively induced 256.71%,270.23%(P<0.05), PAX-2 protein expression level are significantly decreased 71.72%(P<0.05), ki-67 protein expression level have no difference(P>0.05); no treatment group compared to the control group with no significant difference(P>0.05).Conclusions:1 The high expression of AIB1 can promote the growth of MCF-7 cells. On the contrary, the high expression of PAX2 can inhibit the growth of MCF-7 cells.2 The high expression of AIB1 can reduced the sensitivity of MCF-7 cells to TAM. On the contrary, the high expression of PAX-2 can promote he sensitivity of MCF-7 cells to TAM.3 The high expression of AIB-1 can promote the expression of HER-2. However, the high expression of PAX-2 can inhibit the expression of HER-2.4 With over-expression of AIB-1 or PAX-2 did not affect the expression of Ki-67.5 Raising the expression of PAX2 and depressing the expression of AIB1might improve the resistant condition of TAM. Indicates that the PAX2 and AIB1 can be used as predictors of response to endocrine therapy and drug resistant of tamoxifen. So PAX2 and AIB1 might be novel therapeutic targets. |
PDF Full Text Request