The Study Of The Impact Of ANXA3 On The Proliferation And Invasion Of Breast Cancer Cells

Breast cancer is one of the most common female malignant tumors in the world,its incidence increases year by year.In the European and American countries,breast cancer accounts for 25%~30% of female malignant tumors,while its incidence has risen to the first place among the female malignant tumors in our country.To understand the molecular mechanisms is critical for the treatment of breast cancer and the patients' survival.Therefore,it's an important work to find a specific biological marker for breast cancer diagnosis,staging and treatment.Annexins(ANXA)are proteins of a structurally related calciumdependent phospholipid-binding protein superfamily.They are involved in membrane transport and a series of calmodulin-dependent activities happening at the membrane surfaces,including the transport of vesicles,membrane fusion in exocytosis,signal transduction,and the formation of calcium channel.They can regulate the inflammatory response,cell differentiation,and the interactions among cytoskeleton proteins.In the occurrence and development of tumors,ANXA can mediate cell signaling pathways,cell movement,tumor invasion and metastasis,angiogenesis,cell apoptosis,and drug resistance.ANXA3 is a multifunctional protein which is involved in tumor progression,proliferation,apoptosis,and signaling transduction.The changes of ANXA3 expression have an important influence on the tumor development,transfer and drug resistance.However,ANXA3 has different expression patterns in different tumors,suggesting that ANXA3 plays different roles in the development of tumors.For example,an elevated expression of ANXA3 has positive correlation with the drug resistance of ovarian cancer and the development of colorectal cancer and pancreatic cancer,as well as lung adenocarcinoma metastasis and liver metastasis.However,a decreased expression of ANXA3 can promote the occurrence and development of prostate cancer.Therefore,ANXA3 may serve as an ideal target for cancer therapy and a biomarker for studying tumor progression,proliferation,apoptosis and patients' prognosis.However,the role of ANXA3 in beast cancer is not clear.This study will be divided into four parts to explore the mechanism of ANXA3 in the process of proliferation,invasion and metastasis of breast cancer.The first part: The expression of ANXA3 in primary breast cancer tissue will be detected by the immune histochemical method and the correlation between ANXA3 and patients' clinical biological characteristics will be retrospectively analyzed.The second part: The expression of ANXA3 mRNA and protein will be detected through the application of fluorescence quantitative RT-PCR technique and western-blot technique,respectively.The correlation between them and patients' clinical biological characteristics will be analyzed.The cell cycle of breast cancer tissue and normal breast tissue will be detected by the method of flow cytometry,the correlation between the expression of ANXA3 mRNA and protein in breast cancer tissue and the cell proliferation index will be analyzed.The role of ANXA3 in the development and metastasis of breast cancer will be explored.The third part: The effects of ANXA3 on the proliferation and invasion of breast cancer cells will be explored,the gene of ANXA3 will be inhibited by using a technique called RNA interference in breast cancer cells MDA-MB-231,and then the cell cycle and cell apoptosis will be detected by flow cytometry,the migration ability of cells will be tested by cell scratch experimen and the invasion ability of cells will be tested by Transwell experiment.The fourth part: The nude mice subcutaneously transplanted tumor modle will be set up,the effects of ANXA3 on transplanted tumor growth will be observed by drawing tumor growth curve and using vivo imagingtechnology,HE staining analysis,flow cytometry and fluorescence quantitative RT-PCR methods.Part One The expression of ANXA3 and the correlation between ANX-A3 expression and clinical biological characters in primary breast cancerObjective: To explore the clinical value of ANXA3 in primary breast cancer by analyzing the expression of ANXA3 and the correlation between ANXA3 expression and clinical biological characters in primary breast cancer.Method: A retrospective study encompassing 309 breast cancer patients admitted to the Fourth Hospital of Hebei Medical University between January and December 2009 was performed.The patients were all female,aged 22 to76 years old,the average age 47 years.The expression of ANXA3 was determined by the immunohistochemical examination of tissue sections by the MaxVisionTM method.The correlation between ANXA3 expression and prognosis was validated based on age,menstrual status,tumor size,the number of lymph node metastasis,clinical stage,histological grade and molecular subtypes.All the follow-up data was received via telephone follow-up.The patients' DFS and OS were calculated.COX regression model was adopted to improve the multiple factors analysis.Result:1 The difference of positive expression rate of ANXA3 among patients with negative axillary nodes(51.94%),positive axillary nodes1~3(67.59%)and positive axillary nodes>3(73.61%)was statistically significant(?2=11.123,P<0.05).2 According to postoperative histological grade,the ANXA3 positive expression rate of ?grade group,? grade group and ? grade group were31.42%,65.10% and 69.51%,respectively.The difference among them was statistically significant(?2=16.686,P<0.05).3 ANXA3 expression level had no relationship with patients' age,menstrual status,tumor size and clinical stage(P>0.05).The ANXA3 positiveexpression rates of Luminal A type,Luminal B type,HER2 overexpression type and triple-negative type were 58.46%,58.11%,59.46% and 79.66%,respectively.The positive expression rate of ANXA3 in patients with triple-negative breast cancer was significantly higher than that in patients with other types of breast cancer(?2=9.226,P<0.05).4 Postoperative patients were followed-up for 66–78 months,with a median of 71 months.In 96 patients local recurrence or distant metastasis was observed,and their 71-month DFS was 68.93%(213/309).There were 58 dead cases,and their 71-month OS was 81.23%(251/309).Multivariate Cox analysis was performed with the variables including,tumor size,clinical stage,the number of lymphatic metastasis,histological grade,molecular subtypes,ANXA3 expression and other indexes.The multivariate Cox analysis correlated the levels of ANXA3 expression to retrospectively examined DFS and OS.The analysis revealed that the number of lymphatic metastasis and ANXA3 expression were identified as the independent risk factors affecting DFS and OS of patients with breast cancer,respectively.The relative risks of lymphatic metastasis and ANXA3 expression to DFS prognosis were 2.181 and 1.569,respectively,while those to OS prognosis was 2.052 and 1.698,respectively.Conclusion:1 The positive expression rate of ANXA3 in breast cancer has no relationship with patients' age,menstrual status,tumor size and clinical stage.2 The expression level of ANXA3 has correlation with the number of lymph node metastasis,histological grade and molecular subtypes.The positive expression rate increases in the breast cancer patients with positive axillary nodes>3,? grade or triple-negative type.3 The DFS of patients with ANXA3 positive expression is lower than patients with negative expression.There is no statistically significant difference of OS between them,but the OS has a lower trend in patients with ANXA3 positive expression.4 The number of lymphatic metastasis and ANXA3 expression areidentified as the independent risk factors affecting DFS and OS of patients with breast cancer,respectively.5 ANXA3 can serve as a potential biological marker to evaluate the biology behavior of breast cancer and predict the DFS and OS of patients.Part Two The expression and the role of ANXA3 mRNA and protein in the normal breast tissues and breast cancer tissuesObjective: To analyze the expression of ANXA3 mRNA and protein in the normal breast tissues and breast cancer tissues and to explore the role of ANXA3 in the occurrence,development,and metastasis of breast cancer.Method: Clinical samples were collected from the Breast Disease Center in the Fourth Hospital of Hebei Medical University,Hebei,China.81 samples from breast cancer tissues were collected with complete information from April 2015 to October 2015.The patients are female,age 29 ~ 80,the median age 50.Among them,Infiltrating ductal carcinoma 66 cases,invasive lobular carcinoma 4 cases,infiltrating ductal and lobular carcinoma 2 cases,Cribriform carcinoma 1 cases,secretory carcinoma 1 cases,intraductal carcinoma 7cases.In accordance with the AJCC TNM staging(7th,2010),stage?11 cases,stage?50 cases,stage? 19 cases,stage IV 1 case.In order to obtain the normal samples,0.5 cm × 0.5 cm sized breast tissues were randomly collected at the time of routine sampling,from regions located >7 cm away from the cancer tissues.If these samples were confirmed to be normal gland tissues through pathological examination,they were used in the study as samples from normal breast tissues(the control group).The expression levels of ANXA3 mRNA and proteins were determined by fluorescent quantitative RT-PCR and western-blot,respectively,in 81 samples from breast cancer tissues and normal breast tissues.The relationship between the expression level of ANXA3 mRNA and protein and the clinical biological indicators was analyzed.Flow cytometry,used to detect the cell cycle information,analyzed the differences in the cell proliferation indexes between the cancer and normal tissues.In addition,the correlation between the expression levels of ANXA3 mRNA and proteins and the cell proliferation indexes in breast cancer tissueswas calculated.Results:1 The expression levels of ANXA3 mRNA and protein were significantly higher in breast cancer tissues than those in control samples.2 Furthermore,their expression patterns were significantly different in patients with different axillary lymph node metastasis states,with or without vascular tumor thrombus,and with different molecular classifications.3 The expression of ANXA3 mRNA and protein in patients with triple-negative breast cancer was significantly higher than other subtypes.4 The results of flow cytometry revealed that the percentage of G0/1 cells in samples from breast cancer tissues(58.85±9.32%)was significantly lower than the value observed in samples from normal breast tissues(82.52±3.86%;P <0.01).However,the cell proliferation indexes of breast cancer tissues(41.15±9.32%)were significantly higher than the indexes estimated for normal breast samples(17.48±3.86%;P <0.01).5 The ANXA3 mRNA and protein expression levels in breast cancer tissues were positively correlated with the cell proliferation indexes,with the Pearson correlation coefficient as 0.304 for mRNA(P = 0.006)and 0.341 for proteins(P = 0.002).Conclusion:1 ANXA3 mRNA and protein expression levels in breast cancer tissues are significantly higher than those in normal breast tissue.2 ANXA3 mRNA and protein expression level associates with lymph node metastasis status and the presence of vascular tumor thrombus.The expression level of ANXA3 in patients with triple-negative breast cancer is significantly higher than that in patients with other types of breast cancer.3 ANXA3 mRNA and protein expression level of breast cancer tissues is positively correlation with cell proliferation index.4 ANXA3 mRNA and protein expression level of breast cancer tissues has no relationship with patients' age,menstrual status,tumor size and clinical stage and histological grade.Part There The study of the impact of ANXA3 on the proliferation and invasion of breast cancer cellsObjective: To explore the mechanisms involved in the regulatory effects of ANXA3 on proliferation,apoptosis,invasion and migration of breast cancer cells.Method: The expressions of ANXA3 mRNA and protein in high invasive human breast cancer cell line MDA-MB-231 and low invasive breast cancer cell line MCF-7 were determined by fluorescent quantitative RT-PCR and western-blot,respectively.MDA-MB-231 cell line with high expression of ANXA3 was used to the further experiment.Three shRNA plasmids against ANXA3 gene were successfully built and then transfected into MDA-MB-231 cells by the Lipofectamine transfection method.ANXA3-sh2 had the highest inhibition efficiency on ANXA3 mRNA in MDA-MB-231 cells.The stable transfection cells were selected and named MDA-MB-231-Sh.The cells transfected by NC plasmid were named MDA-MB-231-NC.The experiment was divided into three groups: the transfection group transfected by ANXA3-sh2 plasmid,the negative-transfection group transfected by NC plasmid and the control group without transfection.The expression of ANXA3 protein was measured by western-blot.Cell cycle distribution and apoptosis were assessed by flow cytometry.Migration and invasion of the transfected cells were evaluated by using wound healing and Transwell assays,respectively.Results:1 The level of ANXA3 mRNA was significantly higher in the MDA-MB-231 cells than the level in the MCF-7 cells[(0.0696±0.0248)vs.(0.0236±0.0149)](P<0.01).The expression level of ANXA3 protein was significantly higher in the MDA-MB-231 cells than the level in the MCF-7cells[(0.76±0.02)vs.(0.40±0.02)](P<0.01).2 Western-blot showed that the ANXA3 protein level was significantly lower in the transfection group than those in the negative-transfection group and the control group(P<0.01).3 In addition,the percentage of G0/1 cells was significantly higher[(59.27±0.87)% vs.(53.63±1.80)% and(52.40±2.91)%],the apoptosis rate was significantly higher[(6.73±0.33)% vs.(3.58±0.23)% and(3.09± 0.33)%],while the cell proliferation index was significantly lower [(40.67± 0.93)% vs.(46.40±1.85)% and(47.60±2.91)%] in the transfection group when compared with the negative-transfection group and the control group(P<0.05).4 The cell migration and invasion abilities were also lower in the transfection group than these abilities in the negative-transfection group and the control group(P<0.01).Conclusion:1 The levels of ANXA3 mRNA and protein are significantly higher in MDA-MB-231 cells than the levels in MCF-7 cells.2 Three shRNA plasmids against ANXA3 gene are successfully built and ANXA3-sh2 has the highest inhibition efficiency on ANXA3 mRNA in MDA-MB-231 cells.3 By inhibiting ANXA3 expression,apoptosis rate and cell proliferation index of MDA-MB-231 cells decreases significantly,invasion and migration ability increases.ANXA3 has the potential to be an useful biological marker for the diagnosis,treatment and prognostic evaluation of breast cancer.Part Four The impact of ANXA3 on the growth of breast cancer cells in nude mice subcutaneously transplanted tumor modelsObjective: To observe the impact of ANXA3 on the growth of breast cancer cells in nude mice subcutaneously transplanted tumor models and to verify the regulation function of ANXA3 in the proliferation of breast cancer cells.Method: The breast cancer cells MDA-MB-231,MDA-MB-231-sh and MDA-MB-231-NC were prepared and then the tumor-burdened animal models of nude mice were established.18 female nude mice were evenly divided into three groups: the control group,the transfection group and the negative-transfection group,inoculated with MDA-MB-231 cells,MDA-MB-231-sh cells and MDA-MB-231-NC cells,respectively.The spirit,dietand defecation of nude mice were observed and record for 4 weeks since the transplanted tumor began to grow.The weight of the nude mice and the long and short diameter of tumors were measured once a week and then the tumor growth curve was drawn.The degree of growth activity of transplanted tumors was detected by vivo imaging technique.The tissue structure of transplanted tumors was analyzed by HE staining.The cell proliferation and ANXA3 mRNA expression in the subcutaneously transplanted tumors of nude mice were detected by flow cytometry and fluorescence quantitative RT-PCR.The differences among the three groups were recorded and compared.Results:1 The transplanted tumor began to grow after 10 days of inoculation,the rate of tumor formation was 100%.2 The growth curve showed that the growth of transplanted tumors in the transfection group was slower than those in the control group and negativetransfection group.The growth activity of the transfection group was lower than those of the control group and the negative-transfection group.The tumors' mass and volume of he transfection group [(58.55±31.92)mg and(90.62±74.44)mm3] decreased significantly compared with the control group[(222.40±133.40)mg and(239.30±110.90)mm3]and the negative-transfection group [(239.30±110.90)mg and(406.72±95.23)mm3](P<0.01).3 In the control group and negative-transfection group,the tumor cells and vessels were abundant and the mitoses were more common.While in the transfection group,the tumor cells and vessels were relatively scarce and accompanied by significant fibrous connective tissue hyperplasia.4 In the transfection group,the expression of ANXA3 mRNA and protein in the transplanted tumor cells [(0.0071±0.0021)and(0.26±0.02)] and was significantly lower than those in the control group [(0.0671±0.0087)and(0.72±0.04)] and the negative-transfection group [(0.0698±0.0104)and(0.71±0.06)](P <0.01).5 The cell proliferation rate(26.17±3.92)% was significantly lower,while the percentage of G0/1 cells was significantly higher in the transfectiongroup compared with the other two groups(P<0.01).Conclusion:1 After the inhibition of ANXA3 expression in breast cancer cells MDA-MB-231,the growth of transplanted tumors in the transfection group is slower,the tumors' mass and volume decreases significantly compared with the control group and the negative-transfection group.The tumor cells and vessels are relatively scarce in the transfection group.2 The expression of ANXA3 mRNA and protein in the transplanted tumor cells is significantly lower than those in the other two groups.3 The cell proliferation rate is significantly lower,while the percentage of G0/1 cells is significantly higher in the transfection group.4 The proliferation and apoptosis of breast cancer cells can be regulated and the growth of breast cancer cells can be inhibited by silencing ANXA3 expression in breast cancer cells MDA-MB-231.ANXA3 could become a new potential targets for gene therapy of breast cancer.