The Role Of CIP2A In The Development Of Endometrial Carcinoma And The Research Of Its Expression And Function

[Background]Endometrial carcinoma is one of the most common gynecological cancers and the incidence has been increasing.In USA,it was estimated that there were approximate 54870 new cases and approximate 10170 deaths in 2015.Endometrial cancer has been broadly classified into two subtypes(type ?,endometrioid and type ?,non-endometrioid)on the basis of histological characteristics,hormone receptor expression and grade.The most common subtype is low-grade,endometrioid,hormone-receptor-positive endometrial cancer,which has a good prognosis.Type ? endometrial cancers are described as non-endometrioid,high grade,hormone-receptor negative tumours that are associated with a higher risk of metastasis and a poor prognosis.Many patients with endometrial cancer are diagnosed early due to irregular vaginal bleeding at an early stage,and usually have good prognosis,the five-year survival rate were about 85%.Even so,the disease-related death rate is still 5%-15%.The patients with tumor of high-grade and type ? may have poorer prognosis.Although endometrial carcinoma most often affects postmenopausal women,the incidence among younger women has been on the rise in recent years.Therefore,there is an urgent need to research more about the tumorigenesis of endometrial carcinoma,find a new biomarker to more accurately predict survival of Patients.CIP2A(Cancerous inhibitor of protein phosphatase 2A),also named p90 or KIAA1524,is a novel human oncogene.In addition,overexpression of CIP2A has beenfound in various human cancers including breast,prostate,gastric,lung,hepatocellular,colon,and renal cancers,and its expression have good correlation with the occurrence of tumor progression and associated with poor prognosis.The regulation and mechanisms by which CIP2A promotes carcinogenesis has been extensively reviewed recently.Given the relatively low levels of CIP2A expression in normal tissues,this protein is of considerable interest to cancer biologists as a promising drug target.However,the expression and function of CIP2A in endometrial cancer tissue and its involvement in progression of cells were not clear.In this study,we assessed CIP2A expression in 150 cases of primary endometrioid adenocarcinoma tissues,30 cases of normal endometrium tissues,14 cases of endometrial atypical hyperplasia tissues and 20 cases of endometrial hyperplasia tissues by immunohistochemistry,analyzed the differences between different endometrium Ieisions and correlations between the CIP2A expressions and the clinicopathological variables such as age,clinical FIGO stage,histologic grade,etc.Examine the expression of mRNA and protein in fresh tissue of endometrial cancer and normal endometrium by RT-PCR and western blot.Furthermore,in order to clarify the role of CIP2A in endometrial cancer development,we used CIP2AsiRNA in human endometrial carcinoma cell lines ishikawa and An3ca respectively,and then evaluated the effect of CIP2A depletion on cell proliferation,invasion and apoptosis,observed the expression of cell cycle and apoptosis related proteins such as c-Myc,cyclinDl,Bcl-2,etc.In this research,we hope to solve several questions:(1)The expression of CIP2A mRNA and protein in endometrial carcinoma and normal endometrium;(2)The correlation between CIP2A expression and the clinicicopathological parameters;(3)The correlation between CIP2A expression and patient's prognosis;(4)The biological function of CIP2A in the development of endometrioid adenocarcinoma.[Methods]1.Patients and tissue sample.Select resection specimens of 150 cases in Affiliated Hospital of Binzhou Medical University,department of Pathology from January 2009 to May 2014.Select 14 cases of atypical endometrial hyperplasia 20 cases of endometrial hyperplasia and 30 cases of normal endometrium for comparison.All sections were reviewed by two pathologists.2.Immunohistochemistry and analysis of correlation to the clinicalpathologicalvariables.We examined the expression of CIP2A in the 150 endometrioid adenocarcinoma(EAC),14 cases of endometrial atypical hyperplasia(EAH),20 cases of endometrial hyperplasia and 30 cases of normal endometrium tissues samples by immunohistochemical method.All slides were observed and scored by 2 independent investigators,and slides were given a total staining index.The relationship between CIP2A and the clinical pathological parameters was analyzed,as well as the correlation between CIP2A and patient's prognosis?3.The survival analysisThe survival curves were plotted using the Kaplan-Meier method and the differences in overall survival were assessed with the log-rank test.Univariate and multivariate survival data was analyzed using a Cox regression model,which also was used to identify whether CIP2A could be an independent prognostic factor.4.The expression of CIP2A in fresh endometrioid adenocarcnoma and normaltissuesWe detected the level of mRNA and protein in 13 cases of endometrioid adenocarcinoma tissues and 13 cases of normal endometrium tissues by Reverse transcription polymerase chain reaction and Western Blotting,and then compared the difference between them.5.Cell lines culture and transfectionIshikawa and An3ca cell lines were transfected by CIP2A-siRNA and negative control.The level of mRNA and protein of CIP2A were assessed 24h,48h later by RT-PCR and Western blotting.6.Functional analysis of CIP2AsiRNA transfection in cell lines.CIP2A-siRNA transfection were constructed to conform the biological function of CIP2A.Cell migration and invasion assays were performed to assess the effect of CIP2A siRNA transfection on endometrial cancer cells.Cell proliferation and apoptosis were also examined after endometrial cancer cells transfected with CIP2A siRNA using CCK-8,cell colony formation assay and flow cytometry.The detection of cell cycle and cell apoptosis related markers were carried out by western blot.[Results]1.CIP2A was overexpressed inendometrioid adenocarcinomaThe immunohistochemistry results showed that,in CIP2A-positive tissues,the positive stain was detected both in cytoplasm and neuclear.The positive expression of CIP2A in NE,EH,EAH and EAC was found to be 26.7%,40%,42.9%and 87.3%,respectively.The difference between them were statistically significant.In 150 cases endometrioid adenocarcinoma,high level expression of CIP2A was observed in 79 cases,low level expression of CIP2A was observed in 52 cases and 19 cases showed negative expression.Furthermore,we also tested the expression of CIP2A protein and mRNA in 13 cases of endometrioid adenocarcinoma tissues and normal endometrium tissues by Western blot and Real time RT-PCR.The results were consistent with the results from immunohistochemistry.That is to say,CIP2A expression was markedly higher in tumor tissues than in the normal endometrium tissues.2.Association between CIP2A and the clinical pathological variables inEndometrial carcinomaCIP2A expression was positively associated with tumor grade,FIGO stage,the expression of P53 and Ki-67.However,there was no relationship between CIP2A expression and patients' age,myometrial invasion,cervical canal or lymph node involvment and the expression of ER,PR.3.The resulets of survival analysisThe five-year survival rate of 150 patients was 96%.The Kaplan-Meier survival curves showed CIP2A expression have statistical correlation with patients' overall survival.The patients with high expression of CIP2A displayed significantly poorer overall survival.Other characteristics associate with poor prognosis include high FIGO stage,low tumor graed,deeper myometrial invasion and high Ki-67 index.Multivariate analysis revealed that the FIGO stage and tumor grade could serve as independent predictive factors of overall survival.4.CIP2AsiRNA transfection effiency in three endometrial cancer cell linesWe detected the transfection efficiency in endometrial cancer cell lines KLE,ishikawa and An3ca by infecting the CIP2AsiRNA using RT-PCR and western blot.The results of RT-PCR revealed that CIP2A expression levels of protein and mRNA were significantly reduced in three endometrial cancer cell lines,the ransfection efficiency after 24h transfecton was 34%,62%and 68%,respectively.The ransfection efficiency after 48h transfecton was 61%,85%and 82%,respectively.The results of western blot showed that the protein of CIP2A was decreased after CIP2AsiRNA transfection 48h in cell lines of ishikawa and An3ca.So we choose those two cell lines to do the following function experiment because of their high knockout effiency.5.Effects of CIP2A depletion on endometrial cancer cells invasion,proliferation,and apoptosisWhen silenced CIP2A in ishikawa and An3ca,the cell magriation and invasion abilities were significantly decreased.The CCK-8 and colony formation assay showed that the ability of proliferation was reduced when the genes of CIP2A were knocked down in ishikawa and An3ca cell lines.The results of flow cytometry showed the rate of apoptosis were increased compared control in both cell lines group,only ishikawa cell have statistical significance.6.CIP2A depletion downregulates c-Myc,cyclinDl expression in ishikawa andAn3ca cell lines,upregulates caspase-3 in ishikawa cell linesWestern blotting analysis revealed that knockdown of CIP2A decreased the protein levels of cyclin D1 and c-myc expression in both two cell lines,while the levels of P53,Bcl-2 and mTOR were not changed.The level caspase-3 protein was increased in ishikawa while have no change in An3ca.Tthese results suggested that CIP2A induced cell proliferation by cyclin D1 and c-Myc regulation;meanwhile,CIP2A regulated endometrial cancer cell apoptosis through caspase-3 low expression.[Conclusion]1.CIP2A was overexpressed in endometrioid adenocarcinoma tissues and cells,and CIP2A expression had relationship with FIGO stage,tumor grade,P53 and Ki-67 index.2.Kaplan-Meier analysis revealed that high expression of CIP2A were significantly correlated with a shortened overall survival.In addition,high FIGO stage,poor tumor grade,deep myometrial invasion,high Ki-67 index were all associated with a poor prognostis.Multivariate Cox regression analysis showed that high FIGO stage,poor tumor differention not CIP2A overexpression were independent risk factors for prognosis of endometrial carcinoma.3.When silenced CIP2A in cell lines of ishikawa and An3ca,the ability of cell proliferation was decreased and the ability of apoptosis was increased.When the genes of CIP2A were knocked down,the ability of migration,invasion and clone was suppressed,suggesting that CIP2A may play an important role in the prognosis of endometrial carcinoma.CIP2A may become new targets for the treatment of endometrial cancer.4.CIP2A promoted cell proliferation by cyclin D1 and c-Myc regulation;meanwhile,CIP2A regulated endometrial cancer cell apoptosis through caspase-3 low expression.