The Study On Expression Of MAGE-A4 In Breast Cancer Tissues And Breast Cancer Cell Lines And Its Function

Currently, tumor biotherapy represented by tumor immunotherapy has become the fourth therapeutic pattern following surgery, chemotherapy and radiotherapy. In recent years, the development of new tumor vaccine has become one of the focuses in the field of tumor immunotherapy. Among the numerous known tumor-associated antigens, the cancer/testis antigens (CTA) displayed the sepicific expression pattern. They expressed in a variety of tumor tissues but not in normal tissues except the testicular germ cell, and occasionally expressed in the placenta. However, testis is immune privilege organ, which does not cause the specific immune response. Therefore, CTA was a suitable target antigen for specific tumor immunotherapy.As a sub-family of CTA, melanoma antigen gene (MAGE) is a big family which was separated from melanoma cells. MAGE gene encodes tumor-sepicific antigenic peptides which were presented to CD8+ T lymphocytes by HLA-I class I molecules, and therefore inducing tumor sepicific killing. MAGE are highly expressed in various forms of cancer, but not in most healthy adult tissues except for the testis and embryo, therefore is an ideal CTL-mmediated target molecule for tumor specific immunotherapy. The study in-depth to MAGE gene will open a door for the molecular diagnosis of tumors and the cilinical application of tumor immunotherapy.Some studies reported that unlike other MAGE-A family members, MAGE-A4 maybe had different biological characteristics. However, its biological functions and molecular mechanism were still unclear.In our study, RT-PCR (reverse transcription-polymerase chain reaction) was adopted to investigate the expression of MAGE-A4 mRNA expression in 77 benign and malignant breast tumors and six breast cancer cell lines. FLAG-pcDNA3-MAGE-A4 expression plasmid was constructed. MTT assay and colony formation assay were used to detect the effect of overexpression of MAGE-A4 on the proliferation and apoptosis of breast cancer cells. Luciferase reporter assay, RT-PCR, Western blot, colony formation assay and TUNEL assay were used to investigate the effect of MAGE-A4 on the transcriptional activity of p53. Cell fractionation assay was adopted to detect the localization of MAGE-A4 in cells. Immunofluorescence was adopted to detect the co-locolization of MAGE-A4 and p73. Immunoprecipitation experiment was used to examine the physical interaction between MAGE-A4 and p53/p73. SiRNA knockdown against MAGE-A4 was used to investigate the effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells.The main research contents and results were shown as follows:PartⅠThe expression of MAGE-A4 mRNA in breast cancer tissues and breast cell linesObjective: To investigate the ralationship between MAGE-A4 mRNA expression and the clinical parameter/biological behaviors of breast cancer through detecting the expression of MAGE-A4 mRNA in normal breast tissues, pericancerous tissues and breast cancer tissues as well as breast cancer cell lines.Methods: RT-PCR was adopted to detect the expression of MAGE-A4 at the transcription level in normal breast tissues, pericancerous tissues and breast cancer tissues as well as six breast cancer cell lines. Retrospectively analyze was used to analyze the relationship between MAGE-A4 mRNA expression and the clinical/biological behaviors of breast cancer including age of the patients, tumor size, TNM stages, pathological types, histology grades, metastasis of axillary lymph nodes, haemal tube tumor embolus, ER/PR status and HER-2 status.Results:1. Ther was no MAGE-A4 mRNA expression in 77 normal breast tissues.MAGE-A4 mRNA expression was detected in 4 of 77 pericancerous tissues and 33 of 77 beast cancer tissues, respectively, and the positive rate were 5.19% and 42.9%, respectively. 2. The ralationship between MAGE-A4 mRNA expression and the clinical parameter/biological behaviors of breast cancerMAGE-A4 mRNA expression was detected in 15 of 25 patients with the age more than 60 years and in 18 of 52 patients with the age less than 60 years, and the positive rate were 60% and 34.6%, with significant difference (χ~2=4.442, p=0.035). There were no significant differences between MAGE-A4 mRNA expression and other clinical parameter/biological behaviors of breast cancer including tumor size (χ~2=2.301, p=0.316), TNM stages (χ~2=0.421, p=0.517), pathological types (χ~2=1.820, p=0.402), histology grades (χ~2=0.229, p=0.892), metastasis of axillary lymph nodes (χ~2=5.707, p=0.058), haemal tube tumor embolus (χ~2=0.000, p=1.000), ER/PR status (χ~2=0.040, p=0.980) and HER-2 status (χ~2=0.090, p=0.764).3. MAGE-A4 mRNA expression in six breast cancer cell linesMAGE-A4 mRNA was negtively expressed in MCF-7, MDA-MB-231, MDA-MB-157, MDA-MB-453 and MDA-MB-468 cells, but highly expressed in MDA-MB-436 cells.Conclusions:1. MAGE-A4 mRNA was not expressed in normal breast tissues. The positive rate of MAGE-A4 mRNA in pericancerous tissues and breast cancer tissues were 5.19% and 42.9%, respectively, suggesting that MAGE-A4 was tumor specific antigen.2. There were no significant differences between MAGE-A4 mRNA expression and tumor size, TNM stages, pathological types, histology grades, metastasis of axillary lymph nodes, haemal tube tumor embolus, ER/PR status and HER-2 status of breast cancer patients. However, MAGE-A4 mRNA expression was positively correlated with patient's age. The positive rate of MAGE-A4 mRNA in the patients older than 60 years was significantly higher than the patients younger than 60 years (p<0.05).3. Among six breast cancer cell lines, MAGE-A4 mRNA was negtively expressed in MCF-7, MDA-MB-231, MDA-MB-157, MDA-MB-453 and MDA-MB-468 cells, but highly expressed in MDA-MB-436 cells. PartⅡThe construction of FLAG-pcDNA3-MAGE-A4 expression plasmid and its functionObjective: To construct the MAGE-A4 expression plasmid with FLAG tag, and investigate its effects on the proliferation and apoptosis of breast cancer cells.Methods: Nested PCR and gene recombination technique were used to construct the MAGE-A4 expression plasmid with FLAG tag, and MTT assay and colony formation assay were used to investigate the effects of MAGE-A4 overexpression on the proliferation and apoptosis of breast cancer cellsResults:1. FLAG-pcDNA3-MAGE-A4 expression plasmid was successfully constructed and expressed well in cells.2. Colony formation assay showed that after MAGE-A4 transfection and G418 selection, the number of colonies of MCF-7 cells was 2±0, which was significant lower than the control group 25±2 (p<0.05), and the number of colonies of MDA-MB-231 cells was 22±2, which was significant lower than the control group 104±5 (p<0.05).3. MTT assay showed that after MAGE-A4 transfection and ADR (1μM) treatment for 24h and 48h, the cell survival rates of MCF-7 cells were 28.56% and 14.65%, which was significantly lower than the control group (42.55% and 29.74%). After MAGE-A4 transfection and ADR (1μM) treatment for 24h and 48h, the cell survival rates of MDA-MB-231 cells were 69.49% and 20.63%, which was significantly lower than the control group (68.57% and 21.85%).Conclusions1. FLAG-pcDNA3-MAGE-A4 expression plasmid was successfully constructed and positively expressed in cells.2. Exogenous MAGE-A4 could inhibit the proliferation of MCF-7 and MDA-MB-231 cells.3. Exogenous MAGE-A4 could increase the ADR-induced cell death of MCF-7 cells but not MDA-MB-231 cells. PartⅢThe relationship between MAGE-A4 and p53 family membersObjective: To investigate the effect of MAGE-A4 on the transcriptional activity of p53 and the physical interaction between MAGE-A4 and p53 family members, and confirm whether MAGE-A4 plays the role partly or completely via p53 pathway.Methods : Luciferase reporter assay, RT-PCR, Western-blot, colony formation assay and TUNEL assay were used to investigate the effects of MAGE-A4 on the transcriptional activity of p53. Cell fractionation assay and immunofluorescence were used to detect the co-localization of MAGE-A4 and p73 in cells. Immunoprecipitation was used to detect the physical interaction between MAGE-A4 and p53 family members.Results:1. The effect of MAGE-A4 on the luciferase activity of p21WAF1 promotor induced by p53In H1299 cells, the luciferase activity in the control group was 1±0.00; the luciferase activity in the group with 25ng pcDNA3-p53 transfection alone was 18.83±2.16 (p<0.01 as compared with the control); the luciferase activity in the group with constant (25ng) pcDNA3-p53 and guadually increased (100ng, 200ng and 400ng) FLAG-pcDNA3-MAGE-A4 trasfection were 39.25±1.68, 45.07±6.78 and 50.49±4.93, respectively (p<0.01 as compared with the control); the luciferase activity in the group with 400ng FLAG-pcDNA3-MAGE-A4 transfection alone was 1.01±0.05 (p<0.01 as compared with the control).2. The effect of MAGE-A4 on p21WAF1 mRNA expressionIn H1299 cells, p21WAF1 mRNA expression in the group with p53 transfection alon was significantly higher than the control group (p<0.01), and p21WAF1 mRNA expression in the group with p53 and MAGE-A4 cotransfection was significantly higher than the group with p53 transfection alone (p<0.01).3. The effect of MAGE-A4 on p21WAF1 protein expressionIn H1299 cells, p21WAF1 protein expression in the group with p53 transfection alon was significantly higher than the control group (p<0.01), and p21WAF1 protein expression in the group with p53 and MAGE-A4 cotransfection was significantly higher than the group with p53 transfection alone (p<0.01).4. The effect of MAGE-A4 on the colony formation in H1299 cellsIn H1299 cells, the colony numbers with G418 resistance were 96±3, 47±2 and 15±1 in the groups transfected with empty vector, 0.5μg pcDNA3-p53 and 0.5μg pcDNA3-p53/0.5μg FLAG-pcDNA3-MAGE-A4, respectively. There were significant difference among these three groups (p<0.01).5. The effect of MAGE-A4 on the apoptosis of H1299 cellsTUNEL staining experiment showed that the apoptotic rate of H1299 cells was 1%, 6.8% and 19.3%, respectively after transfection with 1μg pcDNA3, 0.5μg pcDNA3-p53 and 0.5μg pcDNA3-p53 plus 0.5μg FLAG-pcDNA3-MAGE-A4, respectively. There were significant difference among these three groups (p<0.01).6. The localization of MAGE-A4 in cellsCell fractionation assay confiemed that the exogenous MAGE-A4 was expressed in both cytoplasm and cell nucleus in H1299 cells.7. The colocolization of MAGE-A4 and p73Immunofluorescence showed that in H1299 cells p73 was located in cell nucleus and part of MAGE-A4 was located in cytoplasm and another part of MAGE-A4 was located in cell nucleus, suggestiong that p73 and MAGE-A4 co-locolized in cell nucleus.8. The physical interaction between MAGE-A4 and p53Immunoprecipitation experiment showed that after co-transfected with MAGE-A4 and p53 plasimids, the immunoprecipitates of p53 contained MAGE-A4 and the immunoprecipitates of MAGE-A4 contained p53.9. The physical interaction between MAGE-A4 and p73Immunoprecipitation experiment showed that after co-transfected with MAGE-A4 and p73 plasimids, the immunoprecipitates of p73 contained MAGE-A4 and the immunoprecipitates of MAGE-A4 contained p73. Conclusions:1. MAGE-A4 could enhance the transcriptional activity of p53.2. MAGE-A4 could enhance the proliferation inhibition role and apoptosis activity induced by p53.3. MAGE-A4 was expressed in both cytoplasm and cell nucleus in H1299 cells.4. p73 and MAGE-A4 co-locolized in cell nucleus.5. MAGE-A4 physically interacted with p53 and p73.Part IV The effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cellsObjective: To investigate the effect of MAGE-A4 siRNA on the apoptosis of MDA-MB-436 cells.Methods : SiRNA knockdown experiment was used to inhibit the expression of MAGE-A4. TUNEL assay and flow cytometry (FCM) and PARP cleavage detection were used to investigate the effect of MAGE siRNA on the apoptosis of MDA-MB-436 cells in response to CDDP.Results:1. The inhibitory efficancy of MAGE-A4 siRNA on the expression of MAGE-A4 in MDA-MB-436 cellsAfter the 48h transfection of MAGE-A4 siRNA, the mRNA expression of MAGE-A4 was significantly inhibited. The inhibitory efficancy were 87.5% and 97.5% after 30pmol and 60pmol MAGE-A4 siRNA transfection.2. The result of MTT assayThe cell survivals of MDA-MB-436 cells were 99.20% and 98.62% after ADR (1μM) treatment for 24h and 48h, respectively. Whereas the cell survivals of MDA-MB-436 cells were 79.44% and 68.10% after CDDP (10μM) treatment for 24h and 48h, respectively.3. The result of TUNEL stainingTUNEL staining showed that after MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 12h and 24h, the apoptotic rate of MDA-MB-436 cells were 9.1% and 2.2%,which were significantly decresed as compared with the control group (24.1% and 10.0%) (P<0.05).4. The result of flow cytometry assayAfter MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 24h, the subG1% were (4.68±0.04)%,which were significantly decresed as compared with the control group (10.88±0.119)% (P<0.05).5. The cleavage of PARP proteinWestern blot result showed that after MAGE-A4 siRNA transfection for 48h and CDDP (10μM) treatment for 24h, the caspase substrate PARP were cleaved. The tario of gray scale was 0.129±0.014, which was significantly decreased as compared with the control (0.549±0.023) (P<0.05).Conclusions:1. MAGE-A4 siRNA has obvious inhibitory effects on MAGE-A4 expression.2. MDA-MB-436 was resistant to ADR but sensitive to CDDP.3. MAGE-A4 siRNA could reduce the apoptosis induced by CDDP in MDA-MB-436 cells, suggesting that MAGE-A4 played a role of tumor suppressor under certain conditions.