Dapk Promoter Methylation And ARHI Protein Expression In Epithelial Ovarian Carcinomas

Ovarian cancer is one of the three most common malignant tumors in female reproductive system and has the highest mortality rate,causes and pathogenesis are still indefinite up to now.ovarian cancer is often asymptomatic at the onset and because a lack of eraly diagnosis index,most patients are final diagnosed with advanced stage,the overall5-year survival rate has not yet exceeded45%.Epithelial ovarian cancer(EOC) comprises85%～90%of all ovarian cancer,studying the molecule mechanism of EOC carcinogenesis and development,looking for new biomarkers and therapeutic targets and improving prognosis are still the hotspot of researchs on ovarian cancer.As a tumor supressor gene,DAPK can positively regulate apoptosis, its expression is lost in multiple human carcinomas due to promotor methylation.Abroad studys reported the rate of DAPK promotor methylation varied from0to67%,there was variance which need to further study.New datas has shown that autophagy is one important tumor supression mechanism of organisms, the inhibition of autophagy may take part in the development of tumor. DAPK and ARHI(aplysia ras homolog1) are both autophagy-ralated tumor supressor genes,to study the exprssion of DAPK and ARHI and their correlation in EOC contributes to know the role of autophagy in the carcinogenesis and progression of EOC.Through detecting DAPK promotor methylation,DAPK protein expression and ARHI protein expression in different ovarian tissues,this research investigate the roles of DAPK and ARHI in the development of EOC,expect to find out new direction for the diagnose and therapy of EOC.ObjectiveTo study the effect and significance of DAPK promotor methylation,DAPK protein expression and ARHI protein expression in the process of initiation and progression of EOC;To discuss the relationship and significance between DAPK promotor methylation and its protein expression in EOC;To analysis the relationship and significance between DAPK protein expression and ARHI protein expression in EOC.Materials and methods1. Study objects135cases paraffin embedded tissues which were collected from the pathology department of The Third Affiliated Hospital of Zhengzhou University from January2009to March2012,were pathologically confirmed containning55cases of epithelial ovarian carcinoma tissues(Malignant Group),25cases of borderline epithelial ovarian neoplasms tissues(Bordline Group)30cases of benign epithelial ovarian neoplasms tissues(Benign Group) and25cases of normal epithelial ovarian tissues(Normal Group).The normal epithelial ovarian tissues origin from the patients undergoing hysterectomy and unilateral or bilateral oophorosalpingectomy because of benign uterine diseases.the age of EOC patients ranged from23to78years,the median age was53years,including8samples of endometrioid carcinoma,12samples of mucinous cystadenocarcinoma and35samples of serous cystadenocarcinoma, they all had not received preoperative radiotherapy and chemotherapy.2. Methods DAPK promoter methylation in aforementioned135cases paraffin-embedded tissue specimens was detected by methylation-specific polymerase chain reaction. Immunohistochemical streptavidin-peroxidase method was also performed to assess DAPK expression and ARHI expression in the aforementioned specimens.3. Statistics approach SPSS17.0statistical software was applied to deal with the data anylasis. The methylation results and immunohistochemical datas are evaluaed through Chi-square test, significant level a=0.05;four-fold table Chi-square test is used to the comparision between two groups, significant level a’=a/comparision times=0.0083. The correlation between gene methylation and its protein epression and the correlation beween two proteins expression was assessed by correlation anylasis of paired datas, significant level a=0.05,contingency coefficient is applied to indicate the correlation degree. Chi-square test is also used to evaluate the relationships of gene methylation and two kinds protein expression with clinical pathological features, significant level a=0.05。Results1. The rate of DAPK promoter methylation was0(0/25),6.7%(2/30),16.0%(4/25),and47.3%(26/55) in normal, benign, borderline, and malignant groups, respectively. This rate was significantly higher in the malignant group than in the three groups (P<0.0083).2. The rate of DAPK-positive protein expression was96.0%(24/25),90.0%(27/30),48.0%(12/25), and30.9%(17/55) in normal, benign, borderline, and malignant groups, respectively. This rate was significantly lower both in malignant and borderline groups than that in benign and normal groups, respectively (P<0.0083).3. The rate of ARHI-positive protein expression was92.0%(23/25),86.6%(26/30),44.0%(11/25), and36.4%(20/55) in normal, benign, borderline, and malignant groups, respectively. This rate was significantly lower both in malignant and borderline groups than that in benign and normal groups, respectively (P<0.0083).4. In EOC,there was negative correlation between DAPK promoter methylation and its protein expression(P<0.001, rp=0.485), there was positive correlation between DAPK protein expression and ARHI protein expression(P<0.001，rp=0.430). 5. In EOC,DAPK promoter methylation and its protein expression are both associated with lymphatic metastasis(P<0.05), but not assocoated with FIGO stage,histo-differentiation and athology category(P>0.05).ARHI protein expression had no correlation with FIGO stage,histo-differentiation, histological type and lymphatic metastasis(P>0.05).Conclusions1. There was moderate methlation of DAPK promoter in EOC, caused gene silencing and inactivity, thereby resulting in downregulation or loss of DAPK protein expression, which contributed to oncogenesis and progression of EOCs.2. As autophagy-ralated tumor supressor gene, the expression of DAPK and ARHI are both downregulated or lost in EOC, they may acted synergistically leading to suprssion of autophagy,and participated in oncogenesis and progression of EOCs together.