Expression Of Receptor Tyrosine Kinase EphA1 And PTK7 In Ovarian Serous Tumors

Ovarian cancer accounting forl/4 of female malignant tumors is one of the most common gynecologic malignant tumors. The mortality rate of ovarian cancer is the first in gynecological tumors. Ovarian serous cancer is the most common histological types of ovarian cancer, accounting for 35% of serous tumors, Benign and borderline tumors account for 50% and 15% of ovarian serous tumor respectively. As ovarian cancer is often asymptomatic in its early stages or lack of reliable biological marker, more than two thirds of the patients with ovarian cancer are diagnosed at advanced stage and the overall survival is very poor. Despite advances in surgery and chemotherapy,5-year survival rate has remained approximately 30%. This situation calls for the investigation of pathogenesis of ovarian serous carcinoma and the identification of molecular markers for early diagnosis and treatment.Receptor tyrosine kinase is one kind of largest transmembrane receptor. It plays an important role in signal transduction, not only involved in the regulation of cell and developmental biology, communication, but also closely related with the occurrence and development of tumor. Eph receptors constitute the largest sub-family of receptor tyrosine kinases. EphAl was the first member of the Eph receptors tyrosine kinase family ever discovered. PTK7 was a new member of receptor tyrosine kinases recently discovered. EphA1 and PTK7 organized into the extracellular domain, transmembrane and intracellular domains constitute. The main differences in the structure had two points:1, EphAl ectodomain contains spherical domain of ligand binding with EphrinAl or EphrinA3. PTK7 extracellular domain was composed of seven immunoglobulin-like loops, and no specific ligand has yet been identified.2, Receptor tyrosine kinase domain of EphAl intracellular domain have catalytic activity, but PTK7 intracellular domain have no catalytic activity. The receptor tyrosine kinase EphAl and PTK7 involved in cell proliferation, differentiation, adhesion, apoptosis, the growth and development and play an important role in the occurrence and development of tumors.The expression of EphAl and PTK7 in tumors are the current research hotspot. Up-regulation of EphA1 was known in various cancers including colon cancer, lung cancer, liver cancer, breast cancer, esophageal squamous cell carcinoma, and reduced expression in human breast cancer cell lines, non melanoma skin cancer specimens Up-regulation of PTK7 was known in various cancers including colon cancer, lung cancer, gastric cancer, and low expression in clear cell renal cell carcinoma. The expression of EphAl and PTK7 in ovarian cancer had only 3 articles retrieved. Herath and Wong et al respectively used RT-PCR method and DASL technology to find the high expression of EphA1m RNAin ovarian cancer tissue specimens. Pejovic et al used RNA probes and found that the expression of PTK7 RNA had a linear downward trend from normal epithelial ovarian, ovarian epithelial of high-risk population to ovarian cancer. The subject is based on these studies. Study on the expression of EphAl and PTK7 gene in ovarian cancer were concentrated in the RNA level. In these studies, the number of ovarian cancer containing other ovarian cancer tissue types such as mucinous carcinoma, are less than 20. There is no report about the role of EphA1 and PTK7 in ovarian serous tumors. ObjectiveIn this study, the plasmid LipofectamineTM 2000 （Lip2000） transfection method was used to explore the effects of EphAl on biological behavior of ovarian serous carcinoma cell lines. The protein of EphAl and PTK7 level in normal fallopian tube epithelial cells and ovarian serous tumors were examined by immunohistochemistry staining. We analyzed the relationship between the EphAl and PTK7 protein levels and clinical pathological characteristics and prognosis of patients with serous ovarian carcinoma. Combining with the relationship between the p53 protein levels and prognosis of patients with serous ovarian carcinoma, we detected EphAl, PTK7, p53 protein level to evaluate the prognosis of patients with ovarian serous carcinoma, and compared their accuracy, in order to find a new index to predict the progress and prognosis of ovarian serous carcinoma. MaterialsAll Human ovarian cancer cell line HO8910 and A2780 were purchased from cell bank of Shanghai and used in the present study.Clinical specimens were collected from the Department of Jingling Hospital, Nanjing Maternity and Child Care Center from January 2001 to January 2013. It consisted of 14 normal fallopian tube tissue samples,6 ovarian serous cystadenoma tumor tissue samples,51 borderline serous ovarian tumors tissue samples and 97 serous carcinoma tissue samples. Formalin-fixed and paraffin-embedded tumor tissues were sectioned at 4 μm thickness. None of the patients received preoperative chemotherapy or radiation therapy. This investigation was performed after approval was obtained from the Ethics Committee of Hospital. Informed consent was obtained from each patient.Methods1 Cell CultureFour-micrometer-thick HO8910 and A2780 were maintained in DMEM medium supplemented with 10% fetal bovine serum in a 5% CO2 and 95% atmosphere at 37℃. According to the requirement of cell library, when cell growth reached 80%-90% confluence, passaged or collected for subsequent experiments after digestion by 0.25% trypsin2 Immunocytochemistry （ICC）For ICC staining, untransfected HO8910 and A2780 cells were grown on glass coverslips until 70% confluence, washed with PBS, and fixed with acetone for 15-20 min. The cells were incubated in 3% H2O2 for 10 min and then the cells were incubated at 4℃ overnight with an anti-EphAl and anti-PTK7 polyclonal antibody at a 1:100 dilution in Antibody Diluent, then washed with PBS. Next the sections were incubated with secondary antibody （Dako, UK） for 30 min at room temperature. Color development was performed with 3,3-diaminobenzidine （DAB） for 2 min. Nuclei were lightly counterstained with hematoxylin.3 Plasmid and cell transfectionThe plasmid pCMV6-GFP was presented as a gift from Jiangsu Provincial Key Laboratory of Molecular Medicine of Nanjing Normal University. The plasmid EphAl-pCMV6-GFP eukaryotic expression vector was purchased from ORIGENE company. HO8910 and A2780 cells were respectively divided into three groups: EphAl transfected group （EphAl-TG）, mock group （MG）, and untransfected group （UTG）. MG and EphAl-TG were transiently transfected with plasmid pCMV6-GFP and plasmid EphAl-pCMV6-GFP with Lipofectamine 2000 respectively, according to the manufacturer’s instructions, and UTG did not receive any treatment.4 RNA extraction and RT-PCR assayTotal RNA was extracted from UTG, MG and EphAl-TG 48h after transient transfection using the Trizol kit, according to the manufacturer’s instructions. RT-PCR used RNA which was from UTG, MG and EphA1-TG The sense primer and anti-sense primer for detection of EphAl were designed according to the EphAl mRNA sequence （GenBank accession number: NM₀05232）. The sense primer is 5’-ATCTTTGGGCTGCTGCTTGG-3’and the anti-sense primer is 5’-GCTTGTCCTCTCGATCCACATC-3’.The PCR products are 127 bp long. The P-actin was used as an internal control （GenBank accession number:NM₀01101.3）. The sense primer is 5’-GAGCTACGAGCTGCCTGACG-3’and the anti-sense primer is 5’-CCTAGAAGCATTTGCGGTGG-3’. The PCR products are 416 bp long. The PCR conditions were 30 cycles of 94℃ 3 min,98℃ 10 s,55℃ 15 s, and 72℃ 1 min, with a final extension at 72℃ for 10 min. The RT-PCR products were analyzed by 1.0% （w/v） agarose gel electrophoresis.5 Determination of cell viability （MTT assay）HO8910 and A2780 cells were seeded in 96-well flat-bottomed plates with 1× 104 cells per well in 100 μL of complete growth culture media, followed by incubation at 37℃ （5%CO2 and 95% air） for 24 hours to allow cell attachment. The cells were then treated with being transiently transfected for 48h.50 μL of 1 x MTT was added into each well for 4 h at 37℃. The excess MTT was then aspirated and the formazan crystals formed were dissolved by 150 μL of DMSO. The absorbance, which was proportional to cell viability, was measured at 490 nm.6 Scratch testAs cells formed monolayer and cell alignment reached more than 60%after transfection for 24h,20μl pipette tip was used to draw line equably with "-" in six orifice plate.After rinsed two times with PBS,cells were cultured in DMEM medium containing without serum for 24 h, then observed and photographed. Cell migration was evaluated with rowing six lines randomly in picture and measuring the relative spacing of cells.The analyse was performed using ImageJ software.7 Immunohistochemistry （Envision method）For IHC staining,4-μm-thick sections were deparaffinized in xylene. The sections were given thermal pre-treatment at 60 ℃ for 2 h. After rehydration through a graded ethanol series, the sections were autoclaved in 10 mM citrate buffer （pH 6.0） at 120℃ for 2 min for antigen retrieval, then cooled to 30℃ and washed with phosphate-buffered saline （PBS, pH 7.2）. After blocking non-specific sites had been blocked with 3% H2O2 for 10 min, the sections were incubated at 4℃ overnight with an anti-EphAl（1:100）, anti-PTK7（1:100）and at 37℃,1h with anti-p53（1:200）, then washed with PBS. The subsequent steps were the same with ICC. Two pathologists independently assessed the immunostained slides, and any differences in the staining scores were resolved by consensus.The percentage scoring of immunoreactive tumor cells was as follows:0 （0%）,1（1-10%）, 2（ll-SO%）, 3（50%-80%） and 4（>80%）. The staining intensity wasexternally scored and stratified as follows: 0（negative）, 1（weak）, 2（moderate） and 3（strong）. A final immunoreactivity score（IRS） was obtained for each of the cases bymultiplying the percentage and the intensity score. Protein expression levels were farther analyzed by classifying IRS values as low（based on an IRS value less than 4）or high（based on an IRS value greater than 4）.8 Statistical analysisCells experiments were repeated diree times. Data were expressed as mean ±standard deviation（mean ± SD）. Results were analysed by two sample independent t-test. Chi-square test（Fisher,s exact test） was used to assese associations of EphAland PTK7 protein expression with clinicopatbological variables. Survival curves were constructed using 化e Kaplan-Meier method and the differences between the curves were compared by the log-rank test. COX proportional hazard regression method were performed to identify the mdependent prognostic factors for overall survival. P-values<0.05（twosided） were considered statistically significant. Sperman correlation analysis was used to investigate whether EphAl and PTK7 are associated with P53. All analyses were performed by SPSS software （version 16.0, Chicago, IL）.ResultsThe expression of EphAl protein was negative in HO8910, and weakly positive in A2780. Cell line HO8910 and A2780 were transiently transfected with EphAl-pCMV6-GFP plasmid. A higher proliferation was observed in HO8910-UTG than in HO8910-MG （P<0.05）, and in HO8910-EphAl-TG （P＜0.01）. A higher proliferation was observed in A2780-UTG than in A2780-MG （P<0.01）, and in A2780-EphA1-TG （P<0.01）. After 24h scratch test results were shown that relative cell spacing of HO8910-EphA1-TG （1348 ±37.20） was significantly higher than of HO8910-UTG （101.83±21.40） and HO8910-MG （707±54.71） （P<0.001, P<0.001）. The relative cell spacing of A2780-EphA1-TG （1177±15.13） was significantly higher than that of A2780-UTG （159.83±42.52） and A2780-MG （463±44.99） （P＜0.001, P＜0.001）.The expression of EphAl was more frequently expressed in normal fallopian tube epithelial tissue （14/14,100%）, in serous cystadenoma （100%,6/6）, in ovarian borderline serous tumors （47/51,92.15%） than in ovarian serous carcinoma （42/97, 43.30%, P<0.05）. EphAl protein expression was associated with clinical stages （P=0.004）, WHO grading （P<0.001）, and MDACC grading （P=0.008） in ovarian serous carcinomas.The patients with negative expression of EphAl protein had a poorer outcome than those with positive expression （P=0.001）.PTK7 protein expression was associated with clinical stages （P=0.038） and metastases （P=0.038） in ovarian borderline serous tumors. PTK7 protein expression was associated with clinical stages （P=0.034）, WHO grading （P=0.004）, MDACC grading （P<0.001） and metastatics （P=0.025） in ovarian serous carcinomas. The survival analysis showed that patients with negative expression of PTK7 protein had a poorer outcome than those with positive expression （P=0.017）.Combined with the p53 protein expression in ovarian serous carcinoma, Sperman analysis showed not correlation between EphAl and p53 （P> 0.05）, a weak negative correlation between PTK7 and p53 （r=-0.219, P=0.045）.The Log Rank test of survival analysis of single factor shows:the prognosis of serous ovarian cancer was relatived to the clinical stages （P<0.001）, MDACC grade （P=0.015）, the WHO classification （P=0.003）, metastasis （P=0.006）, tumor location （P=0.042）, EphA1 （P=0.001）, PTK7 （P=0.017） and p53 expression （P=0.007）, while observed no corrections with the maximum tumor size （P=0.262） and age （P=0.639）. Of the prognosis of early stage, the low stage, the WHO classification levels of 1 and 2, having no metastasis, unilateral tumor, high expression group of PTK7 and EphAl and low expression group of p53 was respectively better than the late stage, the high stage, the WHO classification levels of 3, having a metastasis, the double side parts of the tumors, the low expression group of PTK7 and EphAl and high expression of p53.Cox analysis revealed that clinical stage was the independent factors of prognosis, while the MDACC grading （P<0.001）, the WHO classification, metastasis, tumor site, maximum tumor size, age, EphA1, PTK7 and p53 were not the independent prognosis index （P>0.05）.ConclusionThere was no difference in the expression of EphA1 among normal epithelium of fallopian tubes, benign and borderline serous ovarian tumor, whose expression were more frequent than the malignant serous carcinoma. In normal epithelium of fallopian tubes, benign and malignant serous ovarian tumor, the protein level of PTK7 showed a decreasing trend. Both low expression of EphAl and PTK7 had positive correlation with late clinical stage,high grade of tissues, poor prognosis in serous ovarian cancer, which may become new indicators in the auxiliary diagnosis and evaluating prognosis of patients. EphA1 played a inhibitory effect role in proliferation and migration of HO8910 and A2780 ovarian cancer cell line. A weak negative correlation between PTK7 and P53 suggests that PTK7 may exert the function of tumor suppressor gene in ovarian serous carcinoma by through P53 pathway.