The Expression Of ZEB2 And C-myc Protein In Epithelial Ovarian Cancer And Its Clinical Significance

BackgroundAt present the world each year about suffering from ovarian cancer disease more than two hundred thousand women. Compared with other organs, the incidence of ovarian cancer despite the relatively low, but the mortality rate is very high. According to statistics the analysis of the diseases was informed that ovarian cancer mortality has been ranked fifth. Epithelial ovarian cancer is the most common site of ovarian tumors, accounting for all the malignant ovarian tumors from 85% to 90%. Although in recent years, surgery and platinum based combination chemotherapy, also supplemented by new radiotherapy technology improve, target to therapy, biological therapy combined can improve the patient’s prognosis, but the therapeutic effect of EOC has not significantly improved, but due to the relatively high rate of recurrence of advanced, resulting in ovarian cancer 5 years survival rate has been at the bottom of gynecological malignant tumor. EOC is one of the high mortality rate is the main reason for early diagnosis is relatively difficult. EOC that have been in advanced stage, and these patients 5 year survival rate is less than 30%. Early symptoms are not obvious, the ovarian volume is relatively small attached on both sides of the uterus, have increased the difficulty of finding early screening. At the same time the symptoms of nausea and vomiting, abdominal pain, abdominal distention, urinary frequency symptoms and easy and gastrointestinal diseases confused, so ovarian cancer is often referred to as the "silent killer". Effective early screening or treatment of EOC is particularly important.Epithelial mesenchymal transformation of matter abnormal activation is one of the very important factor in the development of tumor, which is closely related to animal embryonic development and tumor cell biological characteristics and is characterized in that epithelial cells lose their polarity and contact inhibition and acquire a mesenchymal phenotype and is involved in the regulation of EOC proliferation, escape, metastasis and angiogenesis.E box zinc finger binding protein 2 as the core factor of EMT, the E-cadherin gene start E box combined to suppress E-cadherin expression and induces cell EMT, mediated by tumor cell adhesion, cell polarity, cell apoptosis, regulation of oncogenes and maintain micro environmental processes, thereby enhancing the ability of infiltration and metastasis of tumor cells. ZEB2 in many malignant tissues can enhance the invasion and metastasis of tumor cells, such as cervical cancer, thyroid cancer, breast cancer, ZEB2 overexpression may enhance the invasion and metastasis of EOC, but so far ZEB2 in EOC tissues expression reports is relatively less. In recent years, the research of molecular genetics and molecular biology of tumor showed that oncogene activation and anti oncogene inactivation and dysregulation of apoptosis gene is the pathogenesis of many tumors. C-myc gene encoding protein is phosphorylated protein of 62KD, located in the nucleus, with DNA binding ability, its expression can promote DNA replication, cell cycle regulation, cell proliferation and differentiation and other functions. C-myc encoding the protein expression in malignant transformation, progress is likely to promote the EOC process.ObjectivesThe purpose of this study is using immunohistochemical two-step technology through the research of normal ovarian tissues, epithelial ovarian cancer tissue E box zinc refers to binding protein 2 (ZEB2) and proto oncogene c-myc (myc expression level, the analysis of ZEB2 and c-myc expression in various tissues and correlation, analysis of the relationship between the two, and to explore the relationship between them and EOC invasion and metastasis and clinical pathological features.Methods1. sample collectionFrom September 2008 to January 2015, the Third Affiliated Hospital of Southern Medical University Department of pathology archived paraffin embedded tissue specimens of ovarian and Xi’an Alina Biotech Corp to provide a total of 204 cases of ovarian tissue, Of which 191 cases of EOC tissues (32 cases of mucinous carcinoma, 159 cases of serous adenocarcinoma),13 cases of normal ovarian tissues, were confirmed by pathology.191 patients with EOC according to the FIGO clinical staging criteria:137 cases were in stage Ⅰ,28 cases of stage Ⅱ,17 cases of stage Ⅲ,9 cases of stage Ⅳ; lymph node metastasis in 173 cases, lymph node metastasis in 18 cases; 182 patients with no distant metastasis, distant metastasis in 9 cases; 139 cases of T staging of T1, T2 32 cases, T3 20 cases. Between the ages of 22 to 81 years old, which is over 47 years old in 182 cases,9 cases were less than 47 years. EOC of all patients were not receiving preoperative radiotherapy, chemotherapy and biological therapy. The control group of 13 cases of normal ovarian tissue for the same period due to non ovarian disease underwent surgical treatment for normal ovarian specimens.2. ImmunohistochemistryParaffin specimens of normal ovarian tissues obtained in 13 cases,191 cases of EOC tissue organization, serial sections. Paraffin sections of xylene Ⅰ and Ⅱ in the dewaxing 30 minutes and dehydrated with gradient ethanol (100%,90%,80%,70%), flushing pH7.4 PBS solution, dropping PH=6.0 citrate buffer, the slice completely immersed in citrate buffer solution, after boiling for antigen retrieval. Adding a resistance (mouse anti human ZEB2 monoclonal antibody and mouse anti human c-myc monoclonal antibody were purchased from American Abcam) mouse anti human ZEB2 monoclonal antibody and mouse anti human c-myc monoclonal antibody incubated at room temperature for 60 min; After washing with two PBS (anti Shanghai chickai Gene Technology Co Ltd) and incubated at room temperature 60min. Rinsed 3 times with PBS* 3min, DAB color, incubated at room temperature for 3～5min, the control of color color under the light microscope,30 seconds after the termination of distilled water color, Counterstain 1 minute, the differentiation of 1% alcohol and distilled water washing back to blue for 5 minutes, sections were immersed in a graded alcohol (70%,80%,90%,95%,100%) dehydrated 2 minutes, xylene Ⅰ and Ⅱ in the transparent with neutral gum mounting, microscope observation dyeing effect.3. immunohistochemical results of judgmentThe results of determination by double blind method, were determined simultaneously by 2 experienced pathologists independently observe each slice after. Criteria for using a semi quantitative scoring method, each slice randomly under the microscope in 5 high magnification, the calculation of 100 cells/vision, according to the staining intensity and percentage of positive cells was determined. The percentage of positive cells:no positive cells 0,1%～1 minutes,10%～2.50%～3,75%～4 points; staining intensity:colorless 0, pale yellow 1 points, brown for 2 minutes, dark brown for 3 minutes. The 2 grades of product>4 is divided into positive and negative for less than 4.4. statistical analysesAll statistical analyses were performed using SPSS 20.0 software. X2 and Spearman rank correlation test was used in the correlation of ZEB2 and c-myc in EOC tissue and normal ovarian tissues expression and the clinicopathological factors and correlation, P<0.05 (two-sided) for the difference has statistical significance.Results1. ZEB2 protein mainly distributed in the cytoplasm of the cell, occasionally also can appear the nucleus expression, pale yellow to brown particles and normal ovarian tissue with low expression rate. And c-myc protein was mainly localized in the nucleus and cytoplasmic expression in a brown granules, the expression in normal tissue rate lower.2. the expression of ZEB2(1) The positive expression of ZEB2 in EOC tissues (49.2% 94/191) was higher than that in normal ovarian tissue (7.7% 1/13), the difference was statistically significant (P=0.007).(2) The expression of ZEB2 and EOC in the pathological type, the positive expression rate of mucinous adenocarcinoma (75% 24/32) is higher than that of serous adenocarcinoma (44% 70/159), the difference was statistically significant (P=0.001).(3) The expression of FIGO and ZEB2 staging, T staging, lymph node metastasis in relation to Ⅳ,Ⅲ positive expression rate was higher than that of I (69.2% 18/26) to Ⅱ (46.1% 76/165), the difference was statistically significant (P=0.028); The positive expression of T in tumor staging rate of T3>T2>T1 (80% 16/20 VS 62.5% 20/32 VS 41.7% 58/139, P=0.002), the difference was statistically significant; The positive expression with lymph node metastasis rate (72.2% 13/18) higher than those without lymph node metastasis (46.8% 81/173), the difference was statistically significant (P=0.04).(4) The expression of ZEB2 in EOC tissues, and the patient’s age (P=0.536), distant metastasis (P=0.326) has no obvious statistical significance.3. the expression of C-myc(1)The positive expression of C-myc in EOC tissues (53.9% 103/191) was higher than that in normal ovarian tissue (15.4% 2/13), the difference was statistically significant (P=0.007).(2) The expression of C-myc correlated with the degree of differentiation of EOC positive expression in low differentiation adenocarcinoma group (59.7%74/124) was higher than that in well differentiated adenocarcinoma group (43.3% 29/67). The difference was statistically significant (P=0.03).(3) Stage there is a relationship between FIGO expression and C-myc staging,T, in Ⅲ to Ⅳ positive expression rate (73.1% 19/26)was higher than that of Ito Ⅱ (51% 84/165), the difference was statistically significant (P=0.035);the positive expression of T in tumor staging rate of T3>T2>T1 (80% 16/20 VS 62.5% 20/32 VS 48.2% 67/139, P=0.016), the difference was statistically significant.(4) The expression of C-myc in EOC tissues, and the patient’s age (P=0.268), pathological type (P=0.38) and lymph node metastasis (P=0.102), distant metastasis (P=0.182) has no obvious statistical significance. 4. the relationship between ZEB2 and C-myc(1) The expression of ZEB2 and C-myc in EOC tissues by Spearman correlation analysis, both have linear correlation, and positive correlation (correlation coefficient R=0.358,P<0.001).(2) The common expression of T and ZEB2 and C-myc and FIGO stages respectively with linear correlation (P<0.001; P=0.008).Conclusion1. ZEB2 mainly distributed in the cytoplasm of the cell, pale yellow to brown particles, with higher expression rate in EOC tissues, and with tumor FIGO stage, tumor T stage increased, positive expression of the ZEB2 rate also increased. EOC in the positive expression of ZEB2 in lymph node metastasis rate is much higher than that without lymph node metastasis, suggesting that ZEB2 expression of EOC in lymph node metastasis plays a promoting role. According to the above results suggest that ZEB2, as a transcription factor, and its high expression rate of EOCTNM staging and lymphatic metastasis but the expression rate of ZEB2 is higher, the higher the stage of TNM, the possibility of lymph node metastasis is also higher. ZEB2 as a cancer promoting genes play an important role in tumor metastasis, it may become a valuable index to evaluate the development and metastasis of EOC.2. C-myc protein was mainly localized in the nucleus, a brown granules, in EOC tissues with higher expression rate, but also with tumor FIGO stage, tumor T stage, tumor differentiation degree for. With the FIGO stage of the tumor increased, the higher the stage of T, the lower the degree of tumor differentiation, the positive expression rate of C-myc is higher, but with age, lymph node metastasis, pathological type, distant metastases were not related. Thus it can be seen that c-myc expression and EOC tumor TNM stage, T stage, tumor differentiation on the exact, c-myc expression more high tumor differentiation degree is low, the higher the TNM staging, is also expected to become a valuable index in evaluation of EOC stage and T classification.3. ZEB2 and C-myc may be correlated in the pathogenesis of EOC, and FIGO in tumor staging and T staging were significantly correlated. On this basis, to further explore the pathogenesis and screening index of EOC, is expected to become the evaluation of combined detection of EOC in a valuable index.