The Expression Of EPS8 In Ovarian Serous Carcinoma And Ovarian Cancer Cell Biology Behavioy Research

BACKGROUNDOvarian cancer is one of the most common gynecological malignancy, who ranks 3rd in its incidence while 1st in mortality. For lack of early obvious symptoms of patients and specific diagnostic method, more than 70% of the patients have been present with lymph node or the pelvic metastasis. That is an important reason for its poor treatment effect and high mortality rate. So it’s of great significance to find a sensitive tumor marker and potential therapeutic target to enhance the clinical curative effect of the treatment of ovarian cancer.EPS8 (epidermal growth factor receptor pathway substrate 8, EPS8), is a kind of multiple domain signal transduction protein expressing widely, which is highly conserved in evolution, expressing in nematodes, fruit flies, African toad and human being. EPS8 of human being is located in from q23 to q24 of chromosome 12, containing about 821 amino acids at relative molecular weight of 97KD.Researches have shown that EPS8’s expression in embryo formation may be associated with specific neuroectodermaI region, and selective cell proliferation, differentiation and polymorphic response of the body organslO. In 1999, Alvarez found that EPS8 could be contained in the mitogenic signaling pathway11, by which it may be possible to mediate invasion and metastasis of the tumor cells.Many studies show that EPS8 expresses highly in many malignant tumors such as pancreatic cancer, esophageal squamous cell carcinoma,glandular cancer, oral squamous cell carcinoma, cervical squamous carcinoma and colorectal cancer, which is closed related to the development, invasion and chemosensitivity of the tumors.Some experts believe that, through EPS 8 protein detection, as a reference to determine tumor recurrence and prognosis indicators.So, what effect does EPS8 have in ovarian cancer? And what concern do cisplatin-resistance and it have? At present, the domestic and foreign research has not yet been elucidated.This research chooses the paraffin section of the ovarian cancer surgeryand theovarian cancer cells as case study, using immune histochemical methods the real-time fluorescence quantitative PCR Technology (RT-PCR) and western blot to expression of EPS8 in ovarian tissue and ovarian cancer cells.To analyze the correlation between the expression and clinical pathological factors in epithelial ovarian cancer; Secondly, using plasmid pll3.7 as the carrier, p113.7-EPS8 shRNA recombinant plasmid was successfully constructed, which provides a useful tool for further study of the effect of EPS8 gene on the biological behavior of ovarian cancer; Finally, cell line SKOV3 was used as the research object to observe the effect of p113.7-EPS8 shRNA recombinant plasmid after transfection on ovarian cancer cell growth, proliferation of the cell cycle and the change of sensitivity incisplatin, and to further explore the effect of EPS8 gene on the biological behavior of ovarian cancer.OBJECTIVES1.Observe EPS8 expression in ovarian cancer surgery paraffin section.2. Observe the expression of EPS8 in the ovarian cancer cell strains.3.To analyze the of correlation between the expression level of EPS8 proteinand clinical pathology factors of ovarian cancer.4.To construct the p113.7-EPS8 shRNA recombinant plasmid, observe the changes of biological behavior such as ovarian cancer cell growth, proliferation of the cell cycle and the change of sensitivity in cisplatin before and after the transfection of p113.7-EPS8 shRNA.METHODS AND CONTENT1. The express of EPS8 is detected in 76 cases of ovarian serous cystadeno carcinoma, 15 cases of benign ovarian tumor and 15 cases of normal ovarian tissue.2. RT-PCR:With ovarian cancer cell strain SKOV3, A2780,8910, and 8910/PM as the esearch objects, observe the differences in EPS8’s expression at mRNA level.3. Western Blot:Observe the differences in EPS8’s expression in the ovarian cancer cell strains, SKOV3、A2780、8910 and 8910/PM.4. Use the technology of recombinant plasmid to construct the p113.7-EPS8 shRNA ecombinant plasmid and sequence it.5. Select ovarian cancer cell lines SKOV3 of high expression in EPS8 protein, use the siRNA technique to silence EPS 8 protein, and detect the biological behavior changes of effect of p113.7-EPS8 shRNA on SKOV3 cells growth, proliferation, cisplatin sensitivity and so on.RESULTS1. Immunohistochemical results show:the expression of EPS8 is at a high level in ovarian srous carcinoma and located mainly in cytoplasm. In the late stage (Ⅲ+Ⅳ) EPS 8 protein expression in ovarian cancer patients is lower than the percentage of early (Ⅰ +Ⅱ) in patients with ovarian cancer, wherethere is significant difference (38.2%vs.66.7%, P<0.05). High level expression of EPS8 protein in lymph node metastasis of ovarian cancer patients is lower than the percentage of those without lymph node metastasis in ovarian cancer (37.7% vs.65.2%, P<0.05). In addition, the data suggests that there is no significant correlation among the EPS8 protein expression level, pathological type, cell differentiation in ovarian cancer patients (P> 0.05).2.Among these four ovarian cancer cell strains, EPS8 has a higher expression in cisplatin resistant cell strain A2780、SKOV3、8910 and lowest one in cisplatin sensitive cell strains 8910/PM. In a subsequent experiment, select the SKOV3 of high expression in EPS 8 as the research object.3.Use the recombinant plasmid Technology to constructf p113.7-EPS8 shRNA recombinant plasmid and sequencing identified the correct sequence. And the results of Western blot detection show that as the extension of recombinant plasmid transfection time,the expression level of EPS8 protein decreased significant.4.MTT assay detecting SKOV3 cell growth curve showed that the cell growth curve of SKOV3 was slowed down compared with the Empty vector transfection group and the blank control group afte rtransfection of pll3.7-EPS8 shRNA, which indicated that EPS8 decreased expressionhas an antiproliferative effect on growth of SKOV3 cells.5.The results of different groups of cell cycle checked by the Flow cytometry showed that after transfection of p113.7-EPS8 shRNA recombinant plasmid, G0/G1 phase significantly increased, S phase rate decreased significantly, which suggested there was a decrease of EPS8 protein expression, which couldreduce the synthesis of DNA in SKOV3 cells.6.MTT assay detectingthe changes for sensitivity to cisplatin of ovarian cancer SKOV3 cells after transfected with pll3.7-EPS8 siRN Arecombinant plasmid, showed the values of IC50 of blank control group and empty plasmid group, were 32.18 μM and 34.87μM. While after transfected with p113.7-EPS8 shRNA recombinant plasmid, IC50 value of ovarian cancer cell lines SKOV3 was 14.17μM, i.e., after the EPS8 expression wasdecreased, the sensitivity of SKOV3 cells to cisplatin was improved about 2.46 times.CONCLUSIONS1.The expression rate of EPS8 is higher in ovarian serous carcinoma and located mainly in cytoplasm. That the statistical analysis of clinical and pathological factors shows that progress in ovarian cancer having related with EPS8 protein in ovarian cancer.2.The specificity of p113.7-EPS8 shRNA recombinant plasmid decreased the expression of EPS8, pand significantly prevented the growth rate of cell proliferation.3.The specificity of recombinant plasmid of pll3.7-EPS8 shRNA decreased the expression of EPS8, which can cause a block of ovarian cancer SKOV3 cell in G0/G1.4. Tne specificity of recombinant plasmid of pⅡ3.7-EPS8 shRNA decreased the expression of EPS8, and significantly improved the sensitivity of SKOV3 cells to cisplatin.