The Effects And Mechanism Of Annexin A2 On Biological Behavior Of Ovarian Cancer

Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women in the U.S.In 2017,there will be an estimated22,440 newly diagnosed cases of ovarian cancer and an estimated 14,080 deaths in the United States.A major contributor to the high mortality rate is the fact that 70% of women with ovarian cancer initially present with metastases throughout the peritoneal cavity.Over the last two decades,The use of paclitaxel/platinum-based chemotherapy,intraperitoneal chemotherapy and new monoclonal antibodies such as bevacizumab had improved the overall survival rate of early stage patients.Since overall survival remains poor,there is a need for new therapeutic paradigms.Further research is needed to understand how molecular pathways contribute to the development of metastasis,recurrence,and resistance of ovarian cancer to chemotherapeutic agents.The epithelial-mesenchymal transition(EMT)is a highly conserved cellular program that allows polarized,immotile epithelial cells to convert to motile mesenchymal cells.This important process was initially recognized during several critical stages of embryonic development and had recently been increasingly concerned about promoting carcinoma invasion and metastasis by researchers.Annexin A2 as a calcium-dependent,phospholipid-binding protein expressed on various cell types.It is up-regulated in various tumor types and plays multiple roles in regulating cellular functions,including angiogenesis,proliferation,apoptosis,cellmigration,invasion,adhesion,etc.Annexin A2 has also been demonstrated to play an important role in the plasminogen activator system,regulating cytoskeleton structures and actin remodeling which both are essential for tumor invasion and metastasis.We have recently found that both Annexin A2 and plasmin are increased in conditioned media of co-cultured ovarian cancer cells and peritoneal cells,which means Annexin A2 is part of tumor-host signal pathway between ovarian cancer and peritoneal cells which promotes ovarian cancer metastasis.Accumulating evidence suggest that interactions between Annexin A2 and its binding proteins play an important role in the tumor micro environment and act together to enhance cancer metastasis.Recent studies have found that Annexin A2 is related to EMT but the mechanism is unclear.Further investigations of Annexin A2's role and molecular mechanism in ovarian cancer can help to guide the diagnosis and treatment of ovarian cancer.In our study,the expression of Annexin A2,E-cadherin,?-catenin and Ncadherin in epithelial ovarian cancer,benign ovarian tumor and normal ovarian tissue was detected by immunohistochemical staining.This four protein expression trends and the relationship between their expression and clinicopathological features were statistically analyzed,and further analysis was conducted in the four expression of each other in ovarian cancer cases.The expression of Annexin A2 mRNA and protein of human ovarian cancer cell line(SKOV3 and UACC-1598)and human ovarian epithelial cell line(HOSEpiC)were detected by real-time reverse transcription polymerase chain reaction(RT-PCR)and western blot.Then using si RNA silence Annexin A2 expression in SKOV3 and UACC-1598,the proliferation ability was determined by MTT and BrdU,the invasive ability was determined by Transwell invasion assay,the epithelial marker(E-cadherin)and mesenchymal markers(Ncadherin)was detected by western blot.The important role of ?-catenin in the EMT signal pathway induced by Annexin A2 was further analysis.The present study aims to investigate the roles and possible mechanisms of Annexin A2 in ovarian cancer.Part one The expression and relationship with clinicopathology of Annexin A2,?-catenin,E-cadherin,N-cadherin in ovarian cancer ObjectivesThrough the analysis of expression level and trends of Annexin A2,?-catenin,E-cadherin and N-cadherin in normal ovarian tissue,benign ovarian tumor and epithelial ovarian cancer tissues,to find out the relationship between these proteins and clinicopathological of ovarian lesions and staging of ovarian cancer.Methods1.Annexin A2,E-cadherin,N-cadherin and ?-catenin protein level was examined in 40 epithelial ovarian cancers,35 benign ovarian tumors and 30 normal ovarian tissues by immunohistochemical staining.2.Analyzing the expression trends of Annexin A2,E-cadherin,N-cadherin and?-catenin protein,the relationship between epithelial ovarian cancer clinicopathological parameters and the four indicators and the correlation between the four indicators.Statistical methodData were analyzed by SPSS21.0 statistical analysis software.The quantitative variable was expressed by,and comparison between groups using analysis of variance.The comparison of qualitative variable was conducted by test,?=0.05 as the test level and p <0.05 as the criterion of significant difference.Spearman correlation analysis was used to test the correlation between the two variables.Results1.Immunohistochemistry results showed that Annexin A2 protein was expressed on the cell membrane of three ovarian lesions,but the expression intensity in ovarian cancer tissue was significantly higher,the positive expression rate was significantly higher than benign tumor group and normal group(p < 0.05).The positive rate ofAnnexin A2 protein expression in ovarian cancer was not significantly correlated with pathological type(p > 0.05),but the expression level of Annexin A2 protein and ovarian cancer tissue differentiation,clinical stage,lymph node metastasis was significant Positive correlation(p < 0.05).2.Beta-catenin protein was expressed on the cell membrane of three ovarian lesions,but the expression in the normal and benign tumor groups was mainly located in the cell membrane,and the ectopic expression of the cells and cytoplasm was increased in the ovarian cancer tissues.The positive expression rate of ?-catenin protein in malignant group was significantly higher than in benign group and normal group(p < 0.05).The positive expression of ?-catenin protein and ovarian cancer pathological tissue type,tissue differentiation showed no significant correlation(p >0.05).But the expression level of ?-catenin protein and pathological staging,lymph node metastasis was positively correlated(p < 0.05).3.E-cadherin protein was expressed on the cell membrane of normal ovarian tissues,benign ovarian tumors and epithelial ovarian cancers,but the expression intensity in normal tissues and benign ovarian tumors was higher than that of malignant group.The percentage of positive cells on the cell membrane of E-cadherin was less than 70% were considered positive,the positive rate was significantly higher in epithelial ovarian cancer(p < 0.05).E-cadherin protein expression positive rate and ovarian carcinoma pathology type,histological differentiation showed no significant correlation(p > 0.05).However the E-cadherin protein expression positive rate and clinical stage of tumor,lymph Lymph node metastasis was significant correlated(p <0.05),as the stage increased,lymph node metastasis,the expression of E-cadherin protein on cell membrane decreased.4.N-cadherin protein mainly in the interstitial cell membrane appears brown particles.No expression was found in normal ovarian tissues and benign ovarian tumors.The positive rate of N-cadherin protein in ovarian cancer was significantly higher than that in normal,benign group(p < 0.05),and was positively correlated with clinical stage of carcinoma,lymph node metastasis(p< 0.05).5.The expression of Annexin A2 and ?-catenin,E-cadherin was positively correlated in ovarian cancer tissues.There was no significant correlation between theexpression of N-cadherin and Annexin A2.?-catenin was positively correlated with E-cadherin,but was no significant correlation between the expression of N-cadherin and ?-catenin.The expression of E-cadherin was not correlated with the expression of N-cadherin.Conclusion1.Compared with normal ovarian tissues and benign ovarian tumor tissues,there were increased positive expression rates of Annexin A2,E-cadherin,?-catenin and N-cadherin in ovarian cancer specimens,and the difference was statistically significant.2.The positive expression rates of Annexin A2,E-cadherin,?-catenin and N-cadherin in ovarian cancer were correlated with clinical stage and lymph node metastasis.3.Annexin A2,?-Catenin is associated with EMT in ovarian cancer and may plays an important role in the occurrence and development of ovarian cancer.Part two The effects and mechanism of Annexin A2 on the biological behavior of ovarian cancer cellsObjectiveThe purpose of this study was to investigate the effects of Annexin A2 expression on the biological behavior of ovarian cancer cells,to further explore the role of ?-catenin in the function of Annexin A2,to provide a new theoretical basis and experimental data for molecular targeted therapy of ovarian cancer.Methods1.The expression of Annexin A2 in human ovarian cancer cell line SKOV3,UACC-1598 and normal human ovarian epithelial cell line HOSEpi C was detected by West Blot method.2.According to literature Annexin A2 si RNA was synthesised and transfected into ovarian cancer cell lines SKOV3 and UACC-1598.The inhibitory effects of Annexin A2 si RNA on Annexin A2 m RNA and protein levels in ovarian cancer cell lines SKOV3 and UACC-1598 were detected by RT-PCR and Western methods.To explore the influence of Annexin A2 on ovarian cancer cell lines,we conducted the Brd U and MTT assays to measure SKOV3 and UACC-1598 cell proliferation with Annexin A2 inhibition.Transwell method was used to detect the invasion ability of ovarian cancer cell line by inhibition of Annexin A2 expression.Western Blot assay was used to detected the expression of epithelial mesenchymal transition molecular markers(E-cadherin and N-cadherin)of ovarian cancer cells by inhibition of Annexin A2 expression.The expression of total ?-catenin and in the nucleus?-catenin of two cell line SKOV3,UACC-1598 after transfection were detected by Western Blot.3.The eukaryotic expression vector of ?-catenin was successfully constructed and named pc DNA.3.1-?-catenin.Annexin A2 si RNA and pc DNA.3.1-?-catenin were co-transfected into ovarian cancer cell lines SKOV3 and UACC-1598.The proliferation,invasion abilities and EMT of ovarian cancer cells with down-regulated Annexin A2 and up-regulated ?-catenin were detected.Statistical analysisThe data processing is carried out by SPSS21.0.Date was expressed by when they are consistent with the normal distribution.The data are not consistent with the normal distribution and then subjected to normal distribution after processing.Multi-group data was analyzed by the single factor analysis of variance(including LSD Test and Bonferroni test).?=0.05 as the test level and p<0.05 was considered statistically significant.Results1.Expression of Annexin A2 in cell lines SKOV3?UACC-1598?HOSEpi CThe expression of Annexin A2 was detected in all three cell lines,but the expression in ovarian cancer cell line SKOV3 and UACC-1598 was significantly higher than that in normal ovarian epithelial cell line HOSEpi C.The relative protein expression of Annexin A2 in three cell lines was 2.73±0.17,3.22±0.25,0.96±0.13,respectively.There are Significant difference between SKOV3 ? UACC-1598 and HOSEpi C.2.Expression of Annexin A2 m RNA after transfection of Annexin A2 si RNA into ovarian cancer cell line SKOV3 and UACC-1598Cells were harvested after Annexin A2 si RNA transfection for 48 hours,and then detect the inhibitory levels of Annexin A2 m RNA.The non transfected si RNA,transfected non special si RNA and transfected Annexin A2 si RNA were defined as blank group,control group and experimental group.Compared with the blank group and negative control group,Annexin A2 m RNA levels of experimental group were significantly reduced,and the difference was statistically significant(p < 0.01).The relative Annexin A2 m RNA levels in the experimental group,the control group,the blank group of SKOV3 were 1.22±0.02,3.53±0.16,3.37±0.53.The relative m RNA levels in UACC-1598 were 1.08±0.05,3.16±0.28,3.09±0.73,respectively.3.Expression of Annexin A2 protein after transfection of Annexin A2 si RNA into ovarian cancer cell line SKOV3 and UACC-1598Cells were harvested after Annexin A2 si RNA transfection for 48 hours,and then detect the inhibitory levels of Annexin A2 protein.Compared with the blank group and negative control group,Annexin A2 protein levels of experimental group were significantly reduced,and the difference was statistically significant(p < 0.01).The relative Annexin A2 protein levels in the experimental group,the control group,the blank group of SKOV3 were 0.93±0.05,3.36±0.18,3.14±0.16.As the relative protein levels in UACC-1598 were 1.32±0.15,3.42±0.53,3.56±0.22,respectively.4.The proliferation of ovarian cancer cells with Annexin A2 inhibition.The cell proliferation was detected by Brd U and MTT assays in SKOV3 and UACC-1598 with Annexin A2 inhibition.By Brd U assays,the absorbance(OD)values in the experimental group,the control group,the blank group of SKOV3 were0.53±0.08,0.79±0.02,0.82±0.03.As in UACC-1598 were 0.42±0.01,0.88±0.06,0.91±0.06,respectively.By MTT assays,the absorbance(OD)values in the experimental group,the control group,the blank group of SKOV3 were 0.37±0.08,0.69±0.16,0.75±0.12.As in UACC-1598 were 0.42±0.06,0.77±0.15,0.83±0.13,respectively.Compared with blank group and negative group,the proliferation ability of SKOV3 and UACC-1598 cells was significantly decreased after transfection of Annexin A2 si RNA(p<0.05).5.The invasion of ovarian cancer cells with Annexin A2 inhibitionThe average invasive cells by transwell assay in the experimental group,the control group,the blank group of SKOV3 were 46±12,88±9,93±16.The average invasive cells by transwell assay in the experimental group,the control group,the blank group of UACC-1598 were 58±5,85±12,89±10.Compared with the blank cell group and the negative control group,the number of invasive cells in the experimental group was significantly reduced,and the invasive ability of the cells was significantly decreased.The difference was statistically significant(p<0.05).6.The protein expression of E-cadherin and N-cadherin in SKOV3 and UACC-1598 cells with Annexin A2 si RNA transfectionThe protein expression of E-cadherin and N-cadherin after Annexin A2 si RNA transfected into SKOV3 in the experimental group,the control group,the blank group were 4.37±0.88,2.19±0.07,2.08±0.75;1.97±0.39,4.07±0.93,3.96±0.83.The protein expression of E-cadherin and N-cadherin after Annexin A2 si RNA transfected into SKOV3 in the experimental group,the control group,the blank group were1.97±0.39,4.07±0.93,3.96±0.83;1.99±0.21,3.98±0.85,4.35±1.10.Compared with the blank cell group and the negative control group,the expression of E-cadherin in the experimental group was significantly up-regulated,and the expression of Ncadherin was down regulated,suggesting that the phenomenon of EMT in tumor cells were inhibited(p<0.05).7.The ?-catenin expression in SKOV3 and UACC-1598 cells after transfection with Annexin A2 si RNACells were harvested after Annexin A2 si RNA transfection for 48 hours,and then detect the inhibitory levels of ?-catenin m RNA and protein by western blot and PT-PCR.Compared with the blank group and negative control group,experimental group ?-catenin protein and m RNA levels were significantly reduced,the difference was statistically significant.The relative ?-catenin protein and m RNA levels in the experimental group,the control group,the blank group of SKOV3 were 1.47±0.36,3.83±0.56,3.96±0.83;1.63±0.11,3.99±0.12,4.29±0.25.As the relative protein and m RNA levels in UACC-1598 were 2.05±0.12,3.95±0.52,3.88±0.82;2.05±0.12,3.95±0.52,3.88±0.82(p<0.05).8.The nuclear ?-catenin protein expression in SKOV3 and UACC-1598 cells after transfection with Annexin A2 si RNACells were harvested after Annexin A2 si RNA transfection for 48 hours,extracted nuclear protein and then detect the inhibitory levels of nuclear ?-catenin protein.Compared with the blank group and negative control group,nuclear ?-catenin protein levels in experimental group were significantly reduced,the difference was statistically significant(p<0.01).The relative nuclear ?-catenin protein levels in the experimental group,the control group,the blank group of SKOV3 were 0.37±0.02,1.6±0.03,1.8±0.03.As the relative protein levels in UACC-1598 were 0.63±0.06,1.5±0.14,1.2±0.07,respectively.9.Successful construction of eukaryotic expression vector pc DNA.3.1-?-catenin.The test groups were named as Annexin A2 si RNA,non-special si RNA,Annexin A2 si RNA-?-catenin,Annexin A2 si RNA-pc DNA.3.1.The protein expression of Annexin A2 significantly decreased between Annexin A2 si RNA-?-catenin and Annexin A2 si RNA,and that of ?-catenin significantly increased between Annexin A2 si RNA-?-catenin and Annexin A2 si RNA-pc DNA.3.1,Which means the eukaryotic expression vector pc DNA.3.1-?-catenin was successfully constructed and can be used for subsequent experiments(p<0.05).10.The change of proliferation ability after Annexin A2 inhibition and ?-catenin overexpression in the ovarian cancer cell linesAnnexin A2 si RNA-?-catenin,Annexin A2 si RNA-pc DNA.3.1,Annexin A2 si RNA called the experimental group,control group,blank group,respectively.The cell proliferation was detected by MTT assays.The absorbance(OD)values in the experimental group,the control group,the blank group of SKOV3 were0.67±0.12,0.21±0.06,0.25±0.03.As in UACC-1598 were 0.72±0.10,0.28±0.08,0.3±0.07,respectively.?-catenin overexpression obviously abolished the inhibitory influence of Annexin A2 inhibition to the proliferation abilities of SKOV3 and UACC-1598 cells(p<0.01).11.The invasion effect of Annexin A2 inhibition and ?-catenin overexpression in the ovarian cancer cell linesThe average invasive cells by transwell assay in the experimental group,the control group,the blank group of SKOV3 were 85±26,49±12,51±15.The average invasive cells by transwell assay in the experimental group,the control group,the blank group of UACC-1598 were 91±13,50±18,45±9.Compared with the blank cell group and the control group,the number of invasive cells in the experimental group was significantly increased.?-catenin overexpression obviously abolished the inhibitory influence of Annexin A2 inhibition to the invasion abilities of SKOV3 and UACC-1598 cells(p<0.01).12.The protein expression of E-cadherin and N-cadherin in SKOV3 and UACC-1598 cells with Annexin A2 inhibition and ?-catenin overexpressionThe protein expression of E-cadherin in the experimental group,the control group,the blank group of SKOV3 were 2.42±0.25,3.95±0.15,4.32±0.06.The protein expression of N-cadherin in the experimental group,the control group,the blank group of SKOV3 were4.04±0.27,1.987±0.32,2.02±0.16.The protein expression of E-cadherin in the experimental group,the control group,the blank group of UACC-1598 were2.05±0.15,4.42±0.37,4.02±0.51.The protein expression of N-cadherin in the experimental group,the control group,the blank group of UACC-1598 were 3.89±0.44,2.01±0.22,2.43±0.29.Compared with the blank cell group and the negative control group,the expression of E-cadherin in the experimental group was significantly down-regulated,and the expression of Ncadherin was up regulated,suggesting that ?-catenin overexpression obviously abolished the inhibitory influence of Annexin A2 inhibition to the EMT of SKOV3 and UACC-1598 cells.Conclusions1.Annexin A2 gene plays an important role in the proliferation,invasion and EMT of ovarian cancer cells.2.Inhibition of the expression of Annexin A2 gene could significantly inhibit the proliferation,invasion and epithelial mesenchymal transition of ovarian cancer cells.3.The expression in cell and the nucleus content of ?-catenin were significantly decreased by Annexin A2 down-regulation.4.Down-regulation of Annexin A2 and simultaneous up-regulation of ?-catenin can restore the proliferation and invasion of ovarian cancer cells induced by down-regulation of Annexin A2.Annexin A2 may play a biological role through?-catenin.