The Role Of Wip1 In Apoptosis And Invasion Of Ovarian Serous Cystadenocarcinoma Cells And Its Related Molecular Mechanisms

Ovarian cancer is the most common and challenging malignant disease in the female reproductive system,leading to cancer-related death in women.Due to the atypical symptoms at early stage,most women have advanced ovarian cancer at the time of diagnosis,when it has spread to other organs in addition to ovaries.The 5-year survival rate of ovarian cancer is only 30-40%.Despite the continuous improvement of surgery and chemotherapy,most patients suffer from recurrent ovarian cancer and the prognosis is still not satisfactory.Ovarian cancer poses a serious threat to women's life and health.Currently,the pathogenesis of ovarian cancer remains incompletely understood.Apoptosis is one of the main approaches fordeath of human cells,the process of which is subject to strict regulation of the body.Regulatory imbalance can induce excessive cell proliferation or apoptosis,leading to related diseases.Studies have demonstrated that abnormal apoptosis in ovarian cancer cellsplays a key role in the development of ovarian cancer.Wild-type p53-induced phosphatase 1(Wip1),a p53-dependent protooncogene found in1997,is widely involved in regulation of multiple cell signaling pathways,playing an important role in development of various cancers,including ovarian cancer.Wip1 is highly expressed in malignant tumor tissues,such as breast cancer,neuroblastoma andmedulloblastoma,suggesting that Wip1 is closely associated withmalignant cancer.Wip1,a serine/threonine protein phosphatase which is located in human chromosome region 17q23/q24 in the nucleus,is encoded by the gene protein phasphatase magnesium-dependent 1 delta(PPM1D).Wip1 is one member of protein phosatase type 2C(PP2C)family.Studies have shown a closerelationship between Wip1 gene and apoptosis.Manganese(Mn)exposure led to neuronal necrosis of rats,which were subsequently made into rat models in one study.The nerve cell apoptosis rate was detected to be significantly increased,whereas expression of Wip1 in nerve tissue and cells was remarkably decreased.It was also reported that Wip1 expression was significantly higher in nasopharyngeal cancer and renal cancer tissues than in normal tissues.Wip1 silencing led to markedly accelerated apoptosis of these cancer cells.In HeLa cells,Wip1 silencing significantly reduced cell growth and cloning.Nonetheless,increased apoptosis brought about a remarkable increase in expression of p-p38 MAPK,p-p53 and p53 protein.Therefore,we speculate that Wip1 is involved in the inhibition of cancer cell apoptosis.Elevated Wip1 expression exerted an inhibitory effect on apoptosis.To the best of our knowledge,the role of Wip1 in cell apoptosis of ovarian Serous Cystadenocarcinoma and its specific mechanism have not been reported.In the present study,therefore,the expression of Wip1 in ovarian Serous Cystadenocarcinoma was detected by immunohistochemical staining,Real-Time PCR and Western-blot.In addition,the relationship between Wip1 expression and clinicopathological features were analyzed.Small interfering RNA technique,Real-time PCR,Western-blot and Transwellassay were used to study the effect of Wip1 on apoptosis and invasion of SKOV3 cells,thereby elucidating the role of Wip1 in the development and progression of ovarian Serous Cystadenocarcinoma and its mechanisms.Part One Expression of Wip1 in Ovarian Serous CystadenocarcinomaTissue and Its significanceObjective: To explore expression of Wip1 in Ovarian Serous Cystadenocarcinoma and its relationship with clinicopathological features.Methods: We enrolled 80 cases who presented to Gynecological Department ofthe Fourth Hospital of Hebei Medical University and the First Hospital of Hebei Medical Universityfrom September 2012 to December 2014,including 50 cases with ovarian serous cystadenocarcinoma,20 with borderline ovarian serous cystadenoma and10 with pathologically confirmednormal ovarian tissues after a total abdominal hysterectomy with a bilateraladnexectomy due to uterine fibroid.The age of the patients was 45-67 years old,with a median age of 57 years.All patients had no history of chemotherapy or radiotherapy before operation.According to the criteria of FIGO(2014),there were 20 cases with stage ?and ? and 30 with stage ? and ?.Based on histological grade in WHO,the number of well-differentiated,moderatelydifferentiated and poorly-differentiated diseases was 21,7,and 22,respectively.In addition,there was lymph node metastasis in 30 cases,and no metastasis in 20 cases.The specimens were partially put into-196? liquid nitrogen for quick freeze and then transferred to-80? cryogenic refrigerator for storage.The specimens were partially fixed with 10% formaldehyde and embedded in paraffin.Immunohistochemical staining was performed.The expression of Wip1 in ovarian serous cystadenocarcinoma tissues was detected by immunohistochemistry,RT-PCR and Western-blot.The clinicopathological data were also collected to analyze the expression of Wip1 in ovarian serous cystadenocarcinoma and its relationship withclinical staging,lymph node metastasis and histopathological differentiation.Results:1 Immunohistochemistry analysis to detect expression of Wip1 in ovarian serous cystadenocarcinoma tissuesWip1 was expressed in the cytonucleus and part of the cytoplasm ofthe ovarian serous cystadenocarcinoma cells.And it wasn't expressed in normal ovarian epithelium.The positive rate of Wip1 was 68% in ovarian serous cystadenocarcinoma tissues.The positive rate of Wip1 in borderline ovarian serous cystadenoma was 60%.There was a significant difference among the three groups(Fisher's exact probability,P=0.001).2 Real-time PCR to detect expression of Wip1 in ovarian serous cystadenocarcinoma tissuesThe expression of Wip1 mRNA was significantly higher in ovarian serous cystadenocarcinoma(3.1±0.19)than in normal ovarian tissues(P<0.01).Wip1 mRNA expression in the borderline ovarian serous cystadenoma(2.5±0.23)was increased,as compared with normal ovarian tissues,the difference was significant(P<0.01).Wip1 mRNA expression did not differ significantly in ovarian serous cystadenocarcinomaand borderline ovarian serous cystadenoma(P>0.05).3 Western blot assays to determine the expression of Wip1 in ovarian serous cystadenocarcinoma.The expression of Wip1 protein was higher in ovarian serous cystadenocarcinoma(0.81±0.15)than in normal ovarian tissues(0.45±0.22),and the difference was statistically significant(P<0.05).Wip1 expressionwas significantly higher in borderline ovarian serous cystadenoma(0.82±0.19)than in normal ovarian tissue(0.45±0.22)(P<0.05).The expression did not differ significantly in ovarian serous cystadenocarcinomaand borderline ovarian serous cystadenoma(P>0.05).4 The relationship between Wip1 expression and clinicopathological factors of ovarian cancerThe expression of Wip1 in stage?and?(50%)of ovarian serous cystadenocarcinomatissues was lower than that in stage ? and?(80%)(?2=4.963,P=0.026).Wip1 expression was increased incrementally in high(52.38%),moderate(71.43%)and low differentiation(81.82%)groups,and the difference was not significant among three groups(Fisher's exact probability,P=0.118).The expression was notably higher in groups with lymph node metastasis(83.33%),as compared with groups without lymph node metastasis(45%)(?2=8.104,P =0.004)?Conclusion: Wip1 is highly expressed in ovarian serous cystadenocarcinoma and is closely associated with lymph node metastasis,clinical staging and histological grade.It is suggested that Wip1 is involved in the development and progression of ovarian Serous Cystadenocarcinoma,which provides a new theoretical basis for the study of the pathogenesis of ovarian Serous Cystadenocarcinoma.Objective: To investigate the differential expression of Wip1 in different ovarian cancer cell lines,and the cell line with the highest expression of Wip1 was selected,thus laying the foundation for further study of the mechanism of Wip1 in ovarian cancer cell lines.Methods: The expression of Wip1 mRNA and protein in normal ovarian epithelial cells and ovarian cancer cell lines SKOV3,CAOV3,A2780 and ES2 were detected by RT-PCR and Western-blot.SKOV3 was screened out as an experimental cell line.Wip1 gene in ovarian cancer SKOV3 cells was silenced to observe changes in its biological behaviors.Three pairs of siRNA specific to Wip1(Wip1-siRNA-1,Wip1-siRNA-2 and Wip1-siRNA-3)were synthesized in vitro,and a pair of siRNA served as negative controls.SiRNA was transfected into SKOV3 cell line mediated by Lipofectamine2000.The experiment was divided into three groups:(1)control group(SKOV3 cell group)(2)N-siRNA group: negative siRNA-transfected group and(3)experimental group:Wip1-siRNA transfected(1,2 and 3)group.Western blot was used to detect changes in the expression of protein before and after transfection.The most effective Wip1-siRNA-2 was selected for follow-up experiment.Each group set up three parallel wells.Cells in three groups were seeded into 96-well plates for 24 h.Transfection was performed according to instructions.After incubation for 0h,24 h,48h,72 h and at a concentration of siRNA of 20nmol/L,40nmol/L 80nmol/L to SKOV3 respectively,Western blot was used to detect changes in the expression of protein after transfection.The optimal reaction time(48h)of Wip1-siRNA-2 was determined,in addition to the optimal concentration(80nmol/L).Results:1 Real-time PCR to detect the expression of Wip1 mRNA in normal epithelial cells,SKOV3,CAOV3,A2780 and ES2 cell linesThe expression of Wip1 mRNA in SKOV3 cells(3.85±0.23)was significantly higher than that in normal epithelial cells(P<0.01),as in CAOV3(1.73±0.46),A2780(1.89±0.28)and ES2(1.81±0.19)(P<0.05).Part Two Expression,Screening and Silencing of Wip1 in Ovarian CancerCell Lines2 Western blot to determine the expression of Wip1 protein in normal epithelial cells,SKOV3,CAOV3,A2780 and ES2 cell linesA remarkable increase in the expression of Wip1 protein was seen in SKOV3(0.73±0.09),A2780(0.54±0.039)and ES2(1.17±0.08)cells,compared with normal epithelial cells(0.15±0.038),and there was a significant difference(P<0.01).The expression was also markedly higher in CAOV3cells(0.25±0.045)than in normal epithelial cells(P<0.05).In terms of Wip1 expression in SKOV3,CAOV3,AZ780 and ES2 cells,Wip1 expression was detected to be significantly higher in SKOV3 cells than in the other three cells.SKOV3 cells were selected to analyze the relationship between Wip1 and ovarian cancerin follow-up experiment.3 The effect of silencing Wip1 on SKOV3 cellsThe expression of Wip1 in SKOV3 cells transfected with siRNA-2 was proved to be the least.After Wip1-siRNA-2 was transfected into SKOV3 cells,the expression of Wip1 was decreased with the increase in the concentration of Wip1,with the minimal expression at 48 h or at a concentration of80nmol/L.It was demonstrated that Wip1-siRNA transfected SKOV3 cells had the greatest effect at a specific concentration and reaction time(P<0.01).Conclusion: Wip1 was found to be overexpressed in various ovarian cancer cell lines,and the expression of Wip1 in SKOV3 cells was the highest,and Wip1-siRNA transfected SKOV3 cells had the greatest effect at a specific concentration and reaction time,which provide theoretical support for further elaboration of its molecular mechanism.Part Three The Effect of Silencing Wip1 on Apoptosis in Ovarian CancerCellsObjective: To determine the effect of silencing Wip1 on apoptosis in human ovarian cancer SKOV3 cells.Methods: Annexin?/PI staining was used to detect the apoptotic rate of SKOV3 cells in control group,N-siRNA group and Wip1-siRNA(2)group.The expressions of apoptosis-related genes Bax,Caspase-3,Cleaved Caspase-3,P53 and Bcl-2 in three groups were detected by RT-PCR.Bycontrast,the expression of Bcl-2,Bax,Caspase-3,Cleaved Caspase-3,p38 MAPK,p-p38 MAPK,ERK,p-ERK,JNK and p-JNK in three groups were detected by Western blot.In order to confirminvolvement of p38 MAPK signal pathway in the apoptosis of SKOV3 cells,P38 MAPK specific inhibitor SB203580 was supplemented to detect the expression of p38 MAPK and p-p38 MAPK.Results:1 Flow cytometry to detect the effect of silencing Wip1 on SKOV3 cell apoptosis rateAnnexin?/PI staining revealed that the apoptotic rate of SKOV3 cells was significantly higher in Wip1-siRNA group than in control group(P<0.01).Compared with N-siRNA group,the apoptotic rate of SKOV3 cells in Wip1-siRNA group was also notably increased(P<0.01).In addition,SKOV3 cell apoptosis rate did not differ significantly in both control group and N-siRNA group.2 RT-PCR to determine the effect of silencing Wip1 on the expression of apoptosis-related genes in SKOV3 cellsIn this study,the expression of Bax,Caspase-3,cleaved Caspase-3,P53 and Bcl-2 was detected after silencing Wip1.Wip1 silencing resulted in a significant increase in the expression of P53mRNA(2.5±0.034)in SKOV3cells(P<0.01).Neverthelss,Bax/Bcl-2 and cleaved Caspase-3/Caspase-3 ratio were increased(P<0.05).No significant difference was found in the expression of apoptosis-related protein in control group and N-siRNA group.The results indicated that silencing Wip1 could promote the apoptosis of SKOV3 cells.3 Western blot to determine the impact of silencing Wip1 on apoptosisrelated protein expression of SKOV3 cellsIn the present study,the expression of Bax,Caspase-3,cleaved Caspase-3,P53 and Bcl-2 was detected after silencing Wip1.There was a significant increase in the expression of P53 protein(1.02±0.051)in SKOV3 cells(P<0.01).Both Bax/Bcl-2 ratio and cleaved Caspase-3/Caspase-3 ratio wereincreased(P<0.05).No changes in the expression of apoptosis-related protein were observed in control group and N-siRNA group.4 Apoptosis of SKOV3 cells by silencing Wip1 via p38 MAPK signaling pathwayTo gain insight into the effect of Wip1 silencing,we detected the expression of ERK,P-ERK,JNK,P-JNK,P38 and P-P38 in SKOV3 cells.Upon Wip1 silencing,the expression of P-P38 protein was significantly increased(P<0.05),suggesting P-P38 might be involvedin apoptosis of SKOV3 cells.In order to verify involvement of the p38 MAPK signal pathway in the apoptosis of SKOV3 cells,p38 MAPK specific inhibitor SB203580 was added,which revealed significantly reduced p-p38 expression(P<0.01).Conclusion: Silencing Wip1 can promote the apoptosis of human ovarian cancer SKOV3 cells by up-regulating P53,cleaved Caspase-3/Caspase-3 ratio and Bax/Bcl-2 ratio,and by activating P38 MAPK signaling pathway.Part Four The Effect of Silencing Wip1 on Invasion of Ovarian CancerCellsObjective: To investigate the relationship between Wip1 and invasion of SKOV3 cells,and to explore the effects of Wip1 on the invasion and migration of SKOV3 cells.Methods: Scratch test and Transwell chamber method were used to determine the invasion of SKOV3 cells in vitro after Wip1 silencing.The expression of MMP-2,MMP-9,TIMP-1 and TIMP-2mRNA in SKOV3 cells was detected by RT-PCR.The expression of MMP-2,MMP-9,TIMP-1 and TIMP-2 protein in SKOV3 cells were determined by Western blot.Results:1 Scratch test to determine the effect of silencing Wip1 by Wip1-siRNAon invasion ability of SKOV3 cells in vitroCell migration distance was significantly shorter in the Wip1-siRNA group(30.333±3.670)than in control group(59.333±7.685)(P<0.01).Therewas no significant difference between control group and N-siRNA group(P>0.05).2 Transwell chamber method to determine the effect of silencing Wip1 gene by Wip1-siRNAon invasion ability of SKOV3 cells in vitroAfter Wip1-siRNA transfection,SKOV3 cells had remarkably inhibited invasion ability of the basement membrane.The number of SKOV3 cells migrating into the Transwell chamber membrane was(17.167±1.835),which was significantly less than that in control group(31.167±3.43)(P<0.01).No significant difference was noted between control group and N-siRNA group(P>0.05).3 RT-PCR to detect the expression of MMP-2,MMP-9,TIMP-1 and TIMP-2mRNA in SKOV3 cells after silencing Wip1As compared with control group,significant reduction was seen in the expression of MMP-2mRNA(0.51±0.013)and MMP-9 mRNA(0.4± 0.028)in Wip1-siRNA group(P<0.01).The expression of TIMP-1mRNA(0.98±0.015)and TIMP-2 mRNA(0.97±0.029)wasn't significant difference between control group and Wip1-siRNA group(P>0.05).4 Western blot to determine the expression of MMP-2,MMP-9,TIMP-1and TIMP-2 protein in SKOV3 cells after silencing Wip1The expression of MMP-2 protein(0.43±0.049)and MMP-9 protein(0.31±0.052)in Wip1-siRNA group was significantly lower than that in control group(P<0.01).Nevertheless,the expression of TIMP-1 protein(0.9±0.033)and TIMP-2 protein(0.8±0.039)wasn't significant difference between control group and Wip1-siRNA group(P>0.05).Conclusion: Silencing Wip1 inhibits the invasion of human ovarian cancer SKOV3 cells,possibly by inhibiting the expression of MMP-2 and MMP-9.