| Breast cancer is one of the most common female lethal malignant tumor at present. In recent years, breast cancer morbidity increase rapidly because changes of the way of live and work in China, and it has become a serious impact on a public health problem. Ornithine decarboxylase (ODC) is the first and most important rate limiting enzyme in the synthesis of polyamines, which is highly expressed in many human malignant tumors including breast cancer tissues. When MYC and Max formed a complex with E-box (CACGTG), it can activate ODC expression; but when Max and MYC antagonist MAD family members (including Mnt, MAD1, MXI1 and MAD4) formed a complex with E-box, it can inhibit ODC expression. This suggests that there may be a relationship of MYC, MXI1 and ODC.The relationship between ODC G316A polymorphism and breast cancer prognosis is still elusive. The effects of DFMO, an ODC inhibitor, is related with ODC G316A polymorphism or not also need further explaination. Therefore, our research tried to find out whether ODC G316A polymorphism can be a new gene marker of breast cancer prognosis by exploring the function of ODC G316A polymorphism during breast cancer development and prognosis.ObjectiveAnalysis of the survival time of patients with breast cancer of MXI1, C-MYC protein expression and ODC G316A polymorphism. Study the relationship between ODC G316A polymorphism and the expression of MYC, MXI1 protein; and study the relationship between DFMO and the polymorphism of ODC G316 A.Methods1. The expression of MXI1, C-MYC protein and ODC G316A polymorphism and survival time of breast cancer patients:the expression of C-MYC and MXI1 protein in the tissue of breast cancer was detected by immunohistochemistry. The genotyp of ODC G316A for breast cancer tissue was analyzed by nested PCR and capillary electrophoresis (PCR-RFLP). C-MYC, MXI1 protein, ODC G316A gene polymorphism of expression and breast cancer patients’survival time was analyzed by Kaplan-Meier method.2. The relationship between the expression of MXI1, C-MYC protein and ODC G316A polymorphism:stably transfected with MXI1 shRNA recombinant plasmid in breast cancer cell line MCF-7 to silence the expression of MXI1. The stable transfection cell line were identified by quantitative real-time PCR and Western blot; Using taqman probe RT-PCR to detect the change of ODC G316A alleles after transfected cells. Study the affinity of C-MYC and MXI1 protein for ODC G316A allele by chromatin immunoprecipitation (ChIP).3. The relationship between DFMO and ODC G316A polymorphism:Using the MTT method to detect the change of cell proliferation in MDA-435 cells and SK-br3 cells with different concentrations DFMO. Effects of different concentrations of DFMO on two kinds of cells cycle and apoptosis were detected by flow cytometry.Results1. Analysis of the survival time of patients with breast cancer of MXI1, C-MYC protein expression:In the 300 cases of breast cancer patients,92 (30.67%) patients were with C-MYC high expression, while 208 (69.33%) patients were with low expression of C-MYC (contain 65 cases of moderate staining,103 cases of low-staining,40 cases of non-staining). For MXI1 protein,71 (23.67%) patients were with high expression,229 (76.33%) patients were with low expression (contain 78 cases of moderate staining,109 cases of low-staining,42 cases of non-staining). For C-MYC protein, the breast cancer patients with MYC-HEG group have shorter survival time than MYC-LEG patients, there is a statistically significant difference (χ2=16.003, P=0.000).For MXI1 protein in breast cancer patients, MXI1-LEG group have a shorter survival time than MXI1-HEG group, there is a statistically significant difference (χ2= 7.266, P= 0.007).2. Analysis of ODC G316A polymorphism and survival time of breast cancer patients:In the 300 cases of breast cancer patients,168 patients (56%) died, of which 72 cases (42.86%) were in the ODC GG group, while the others (57.14%) were in the ODC AA/AG group. Further data analysis revealed that 10-year survival rate of patients with ODC GG group was 53.85%, while that was only 33.33% in ODC AA/AG group, the difference was statistically significant (P= 0.000). Our data showed that ODC AA/AG group had worse prognosis than ODC GG group. To further study, we analyzed the relationship between ODC G316A genotyping and staging of patients.There was no significant difference in survival time for patients with stage I breast cancer between ODC GG group and ODC AA/AG group (χ2=0.021 P=0.884), and that was same for stage II breast cancer patients (χ2=0.423 P=0.5125). However, for patients with stage III breast cancer,10-year survival rate of patients with ODC GG group was 44.23%, significantly higher than the 24.00% ODC AA/AG group of patients, the difference was statistically significant (χ2=5.464, P=0.019)3. The relationship between the expression of MXI1, C-MYC protein and ODC G316A polymorphism:The results showed that the high expression rate of C-MYC protein in the ODC AA/AG group was obviously higher than that of ODC GG group (χ2= 17.812, P=0.000). But the low expression rate of MXI1 protein in the ODC AA/AG group is higher than the ODC GG group (χ2=5.524, P= 0.019); When MXI1 gene was silenced, ODC gene and protein expression had no obvious effect, but the expression of A allele of ODC G316 A was higher than G allele. At the same time, Western Blot demonstrated that C-MYC and MXI1 both expressed in breast cancer cell lines MDA-435 and MCF-7 cell lines. And further chromatin immunoprecipitation showed that C-MYC protein and MXI1 protein both bound to ODC G316A allele A and not to allele G4. The relationship between DFMO and ODC G316A polymorphism:In the experiments, we found that the proliferation inhibition of SK-br3 (ODC AA) was stronger than MDA-435 (ODC GG) cell lines after dealt with DFMO:The growth inhibition rates of MDA-435 and SK-br3 dealt with 10mmol/l and 20mmol/l DFMO after 48h were 24.1% and 33.6%,46.3% and 53.5%, respectively, and the differences of OD value have statistical significance (t= 2.134, P=0.021; t=2.213, P=0.019).The growth inhibition rates of MDA-435 and SK-br3 dealt with 10mmol/l and 20mmol/l DFMO after 72h were 28.9% and 35.7%,54.3% and 65.4%, respectively, and the differences of OD value have statistical significance (t=2.434, P=0.015; t=2.489, P =0.013).Respectively measured the apoptosis rate of MDA-435 (ODC GG) and SK-br3 (ODC AA) cells both dealt with 20mmol/l of DFMO after 24h,48h and 72h by flow cytometry, the results were 7.58%±2.06% VS 13.88%±3.45%(t= 2.047, P=0.041),43.28%±14.28% VS 59.96%±16.42%(t=3.680, P=0.000), 77.87%±30.25% VS 93.08%±32.15% (t=3.293, P=0.0001), there were statistically significant differences (P<0.05). Then we measured the cell cycles under the same condition, and the rates of S stage in MDA-435 (ODC GG) and SK-br3 (ODC AA) cells were respectively 13.25%±2.38% VS 12.89% ± 2.21%,21.43% ±3.12% VS 12.24%±3.55%,16.32%±3.23% VS 15.24±3.01%, there was statistically significant differences (t=2.638, P= 0.012) in 48h, while there were no statistically significant differences in 24h and 72h. We can see that after the treatment of breast cancer cell line MCF-7 (ODC AG) by DFMO, the expression of ODC G316A allele A reduced (t=3.708, P= 0.000), and the expression of G had no significant changes.Conclusions:1. High expression of C-MYC and low expression of MXI1 protein can be used as independent poor prognostic factors in breast cancer patients. For ODC G316A polymorphism, the prognosis of patients with ODC genotype of AA/AG genotype in breast cancer was worse than the prognosis of ODC patients with GG genotype. ODC G316A polymorphism may become a new genetic marker for breast cancer patients.2. MXI1 maybe play a regulatory role in the development of breast cancer through binding to the A allele of ODC G316 A.3. The proliferation inhibition and apoptosis to breast cancer cells by DFMO is different in different genetic type of ODC G316A in breast cancer cells, we speculated that possible mechanism of this effect is that DFMO may selectively bind to the ODC G316A allele A, but it need further functional analysis to test. |
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