| Ovarian cancer is one of the most common malignant tumor in females, which ranks first in mortality rate.Ovary hides in the pelvis, because of a lack of early diagnosis, 70% ~ 80% is already advanced stage, extremely easy to metastasis and diffusion, yet even after complete cytoreductive surgery, the recurrence rate is high,the five year survival rate is still hovering around 30% to 40%. Epithelial ovarian cancer has become a malignant tumor which is a serious threat to the lives of women.The full name of PITX2 is "pituitary homeobox 2", which is located on chromosome 4q25-26 and is the member of the same type of box Bicoid gene family,which was named as the origin of the function in pituitary development and plays an important role in the development of vertebrate pituitary gland,brain, heart and teeth. Several studies in recent years have reported that mutation of PITX2 is associated with many diseases, such as Riegel syndrome, iris hypoplasia,and sporadic Peter syndrome,but more and more evidence indicated that abnormal activation of PITX2 in the genesis had a relationship with development of many tumors, such as:colorectal cancer, prostate cancer, breast cancer, thyroid tumor and so on.A large number of studies have shown that the Wnt signaling pathway plays an important role in the occurrence and development of epithelial ovarian cancer. In2002, in pituitary adenomas,Kioussi’s studies have shown that when Wnt signaling pathway is activated, combined with the Wnt protein and cell surface Fz receptorsand co receptors, thereby inhibiting the formation of β-catenin-Axin-APC-GSK-3βcomplex which lead to the inactivation of GSK-3β, β-catenin cannot be GSK-3phosphorylation and degradation, gradually accumulated into the nucleus, with a combination of transcription transcription factor TCF/LEF, initiating transcription of the corresponding downstream target genes PITX2,so as to promote the expression of PITX2 downstream target genes such as C-myc, Cyclin D1, Cyclin D2,the abnormal proliferation of cells are activated, and ultimately promote the occurrence of tumors.PITX2 is a downstream gene of Wnt signal pathway, and is regulated by Wnt/Dvl/β-catenin, but the abnormal activation of PITX2 in epithelial ovarian cancer is still not clear whether is related to Wnt/β-catenin signal pathway.ObjectiveIn this study, we analyzed PITX2 and β-catenin from the tissues and cell lines in order to understand the role of PITX2 in epithelial ovarian cancer and if the overexpression of PITX2 in epithelial ovarian cancer was regulated by Wnt/β-catenin.Materials and methods1.Study objects(1.1)Clinical specimens Surgical specimens were collected in the Third and the First Affiliated Hospital of Zhengzhou University hospital form September 2012 to December 2014, patients aged 16 to 70 years old, the median age was 54 years old, according to the degree of the benign and malignant tissue samples were divided into 3 groups: 42 cases of epithelial ovarian cancer(malignant group);37 cases of benign ovarian tumors(benign group), 35 cases of normal ovarian tissues(normal group); these tissue specimens were fixed by 10% formaldehyde, embedded in paraffin. In 42 cases of ovarian epithelial carcinoma patients, 37 cases of benign epithelial ovarian tumor, 35 cases of non epithelial ovarian tumor patients serum samples, sampling time is 6 am to 8 am, static blood 20 min at the room temperature,were centrifugal 20 min at 4 DEGC 3000 r / min; placed the supernatant in the EP tube;preserved the temperature of-80 DEGC refrigerator. All cases were not associated with other cancers and were not received radiotherapy or chemotherapy as well asafter surgery, were drawn before the communication with patients and their families and to obtain informed consent. All cases were confirmed by pathology after surgery.(1.2)cell lines Two cell lines of SKOV3 and OVCAR3 from the ATCC in USA,culture medium were used in 1640 two cell lines(containing 10% fetal bovine serum,penicillin 100U/ml,fire,100mg/L streptomycin), at the temperature of 37.5 DEGC,5%CO2 incubator, Wnt signaling pathway inhibitor DKK130 ng/ml were added when the cells entered the logarithmic phase.Cell groups:SKOV3 blank control group,SKOV3+DKK1 inhibitor group,OVCAR3 blank control group,OVCAR3+DKK1inhibitor group.2. Methods(2.1). SP method of immunohistochemistry and real-time fluorescence quantitative PCR(Q-PCR) method were used to detect the protein and m RNA expression of PITX2 and β-catenin in different ovarian tissues.(2.2). Elisa was used to measure the protein expression of PITX2 in serum of patients with different diseases of ovary.(2.3).The MTT and cell scratch test were used to assay the cell proliferation and migration ability of SKOV3 and OVCAR3.(2.4). Q-PCR and Western bolt were used to detect the protein and m RNA expression of PITX2 andβ-catenin in cell lines of SKOV3 and OVCAR3.3.Statistics approach SPSS17.0 statistical software was used to analysis data.The Chi-square test was used to compare the positive rates of PITX2 and β-catenin protein in epithelial ovarian tissue and the relationship between two proteins and different clinical pathological parameters.The single factor analysis of variance was used to compare the m RNA expression of PITX2 and β-catenin in different ovarian tissues, between the two two groups were compared by LSD-t method. Pearson coefficient of correlation was used to analysis between the protein expression of PITX2 and β-catenin in epithelial ovarian cancer tissue. Between the two independent samples were compared by t-test.significant level α=0.05, the comparision between two groups, significant level α′=α/comparision times=0.0167Results1.According to the results of immunohistochemical staining showed positive expression of PITX2 protein was located in cytoplasm. The rate of positive expression of PITX2 protein in normal group, benign group and malignant group,was 14.3%(5/35), 16.2%(6/37), 83.3%(35/42)respectively. The positive expression in malignant group was significantly higher than that in normal group and benign group(P<0.0167), normal control group and benign group was not significantly different(P>0.0167).2.In epithelial ovarian cancer tissue, the expression of PITX2 protein in epithelial ovarian carcinoma was related to FIGO staging, differentiation degree and lymph node metastasis(P<0.05); and were not correlated with age(P>0.05).3.The expression of PITX2 protein in the serum of the patients in normal group,benign group and malignant group, was 7.25± 2.24 ng.m L-1, 7.83 ±1.9 ng.m L-1,12.18 ±2.38 ng.m L-1 respectively. The expression of PITX2 protein in the serum in malignant group was significantly higher than that in benign group and normal group,the difference was statistically significant(P<0.01), there was no significant difference between the normal group and the the benign group(P>0.05).4.The relative amount expression of PITX2 m RNA in normal group, benign group and malignant group was 0.128±0.087, 0.167±0.106, 0.405±0.126, respectively.PITX2 in the malignant group the relative expression level of m RNA was significantly higher than that in normal group and benign group(P<0.001), but no significant difference between the normal group and benign group(P>0.05).5.According to the results of immunohistochemical staining,the positive expression of β-catenin protein was in cytoplasm. The positive expression ofβ-catenin protein in normal group, benign group and malignant group, the rate was5.7%(2/35), 10.8%(4/37), 85.7%(36/42), respectively. the positive expression in malignant group was significantly higher than that in normal group and benign group(P<0.0167), normal group and control group showed no significant difference(P>0.0167).6.In epithelial ovarian carcinoma, ectopic expression of β-catenin protein has related to the FIGO stage, histological differentiation and lymph node metastasis(P<0.05); and were not correlated with age(P>0.05).7.The relative amount expression of β-catenin m RNA in normal group, benign group and malignant group were 0.110±0.082, 0.132±0.095, 0.286±0.106, in malignant group, the relative expression level ofβ-catenin m RNA was significantly higher than that in normal group and benign group(P<0.001), but between the normal group and control group no significant difference(P>0.05).8.The ectopic expression of PITX2 protein and β-catenin protein in epithelial ovarian cancer tissue is associated, and between the two were positively correlated(rp=0.548, P=0.003).9.When the Wnt signaling pathway inhibitor DKK130ng/ml were added in SKOV3, OVCAR3 cells, cell proliferation and migration ability were decreased in the cell experiment(P<0.05).10.When the Wnt signaling pathway inhibitor DKK130ng/ml were added in SKOV3, OVCAR3 cells, the expression of PITX2 andβ-catenin m RNA were decreased in the cell experiment(P<0.05).Conclusions1. The abnormal high expression of PITX2 and β-catenin in epithelial ovarian cancer, has related to FIGO stage, histological differentiation and lymph node metastasis, which show that overexpression of PITX2 and β-catenin may be related to the occurrence and development of the epithelial ovarian carcinoma.2. The high expression of PITX2 protein in the serum of patients with epithelial ovarian cancer, suggesting that PITX2 may serve as a good indicator of early diagnosis and evaluation of disease progression in patients with epithelial ovarian cancer.3. PITX2 is involved in the occurrence and development of epithelial ovarian cancer, and PITX2 may act as a downstream target genes in the Wnt/β-catenin signaling pathway,which is regulated by Wnt/β-catenin. |
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