Expression And Significance Of Adenomatous Polyosis Coil Protein And Promoter Region Methylation In Cervical Squamous Cell Carcinoma

1 Background and ObjectiveCervical cancer is a common malignant tumor as the most common malignancy among women worldwide. The molecular biology research has indicated that High-risk human papilloma virus (HPV) infection is an important pathogenic factor in cervical cancer, HPV infection is the basis for cervical cancer. Combined with other factors such as sexual misconduct, productive, poor economic conditions and so eventually cervical cancer appears. Though the above factors play an important role in the pathogenesis of cervical cancer, the exact etiological factors for cervical cancer are not very clear; and there are lack of useful reagents for chemoprevention, sensitive biomarkers for early detection and high-risk subject screening.From the biological point of view, there were multiple genes which were abnormal expression in the process of development of cervical cancer. Tumor suppressor gene in cancer that occurs during the methylation of protein expression or abnormal inactivation is an important mechanism of the development of tumor. It helps to understand the mechanism of tumor by learning more about tumor suppressor gene methylation. Our group who have been through the use of pre-weak cation bead chip (MB-WCX) and matrix-assisted laser desorption ionization time of flight mass spectrometry with (MALDI-TOF-MS) detection of cervical squamous cancer patients and normal control group, the changes in serum proteomic features and laws, established a diagnosis of cervical squamous cell carcinoma of the protein group classification model of cervical cancer screening changes that might be closely related to the candidate proteins. Obviously, on the base of analyzing proteomic changes in cervical squamous cell carcinoma, based on immunohistochemical and methylation technology to further analysis of these proteins in cervical tissue, it will be able to understand the specific proteins and genes in the role of tissue and serum in cervical cancer patients more fully. Therefore, the study uses the tumor suppressor gene called adenomatous polyosis coil (adenomatous polyosis coil, APC) gene which was significant differences in the protein peak in serum between cancer and normal control group, and further to verify and analyze it in cervical cancer tissue by immunohistochemistry and methylation, to explore the correlation between APC and cervical squamous cell carcinoma and provide important information for research on molecular mechanisms involved in cervical squamous cell carcinoma and looking for sensitive and specific indicator and method for early diagnosis and biological control of cervical cancer.2 Materials and Methods2.1 MaterialsCervical cancer specimens were obtained from Obstetrics and Gynecology outpatient surgery or biopsy of the fresh specimens in the First Affiliated Hospital of Zhengzhou University from October 2008 to December 2009, and all organizations were confirmed by pathological examination, pathological types were all the squamous cell carcinoma. It contained 60 cases of cervical cancer which were staged according to FIGO (2000),27 cases were stageⅠ,24 cases were stageⅡ,9 cases were stageⅢ/Ⅳ; 13 cases with lymph node metastasis; histological grade (WHO): 39 patients were grade G1-G2,21 cases were grade G3. The median age of the patients was 45 years old from25 to 65.15 cases of normal cervical epithelial tissue, which were obtained from the patients who operated for uterine leiomyoma at the same time, were collected for the study as control (cervical appearance was smooth, TCT was reported as normal or inflammation before surgery). The patient without any treatment before surgery, and the postoperative pathological data was integrity.2.2 Tissue processingThe fresh tissue was divided into two portions. One was fixed into buffer formalin embedded in paraffin for immunohistochemistry, the other was quickly frozen in liquid nitrogen and then transferred to -80℃refrigerator storage for the methylation-Specific PCR (methylation-Specific PCR, MSP).2.3 MethodsIn this study, we used ABC method to detect APC protein expression; we used MSP to detect the promoter methylation in APC gene, and we also analyzed their combination with the clinical data.2.4 Statistical analysisWe used SPSS 16.0 statistical software to analyze the data, the Continuity Correctionaχ2 test, Pearsonχ2 test, Spearman correlation test were used for data analysis, takingα=0.05 as the test level.3 Results3.1 The expression of APC protein3.1.1 The expression of APC protein in cervical squamous cell carcinoma and normal cervixThe negative expression of APC protein in cervical squamous cell carcinoma was 53 cases (88.33%,53/60) while 1 case in normal cervix (6.67%,1/15), and the difference was significant between the two group (χ2=35.751, P=0.000).3.1.2 The association between the clinicopathological features of cervical squamous cell carcinoma and APC protein expressionIn cervical squamous cell carcinoma, with increasing clinical stage the negative rate of APC protein expression increased:81.48%,91.67%,100%; Histological G1-G2 and G3 was 89.74%,85.71%; with lymph node metastasis than those without lymph node metastasis(92.31% vs.84.21%), these differences were not statistically significant(P value was 0.167,0.966,0.791).3.2 The status of APC gene promoter methylation3.2.1 The status of APC gene promoter methylation in cervical squamous cell carcinoma and normal cervixThere was no APC gene methylation amplified band in 15 cases of normal cervical epithelium specimens, but 65% (39/60) of cervical squamous cell carcinoma had the promoter methylation in APC gene, and the positive rate of methylation of the two groups was statistically significant difference(x2=20.312, P=0.000).3.2.2 The association between the clinicopathological features of cervical squamous cell carcinoma and APC gene methylationIn cervical squamous cell carcinoma, the APC gene promoter methylation positive rate respectively was 74.07% (20/27),45.83% (11/24),88.89% (8/9) in stageⅠ,ⅡandⅢ/Ⅳ, and three positive rate of methylation was significant (P=0.025). The positive methylation rate of APC gene was 58.97% (23/39),76.19% (16/21); in grade G1-G2 and G3, and the difference was not significant (P=0.182). APC gene promoter methylation positive rate was 53.85% (7/13) and 34.21% (13/38) in 13 cases of lymph node metastasis and 38 patients without lymph node metastasis in cervical squamous cell carcinoma cases, and the difference between the two groups was not significant(P=0.211).3.3 The correlation between the APC protein expression and gene methylation in cervical squamous cell carcinomaIn methylation positive and methylation negative of cervical squamous cell carcinoma samples, the rate of reducing or missing in APC protein was 97.44% (38/39),71.43% (15/21), and the difference was significant(P=0.000). We analyzed the information on the association between the two groups, the results indicated that APC gene promoter methylation and APC protein expression was negatively correlated (r=-0.38, P=0.000).4 Conclusion4.1 APC gene promoter methylation was the important mechanism and might be involved in the development of cervical squamous cell carcinoma.4.2 APC gene methylation was a frequent occurrence of cervical cancer incidents.4.3 APC gene promoter methylation abnormalities which may be one of the important reasons that lead to gene transcriptional inhibitory and protein abnormal expression. It may be one of pathogenesis of cervical cancer that in the cervical cancer APC gene promoter hypermethylation leads to low protein expression.4.4 APC gene may play a tumor suppressor gene function, the gene methylation may cause cancer with the tumor development and external factors.