| Background and ObjectiveCervical carcinoma is the second most frequent gynecological malignancy among women worldwide, with highest incidence rates in developing countries, and it has a younger trend in recent years. It is estimated that 490,000 new cervical carcinoma cases are diagnosed and 270,000 deaths will occur in worldwide annually. Infection with human papillomavirus (HPV) is the most significant risk factor in its etiology, but only HPV infection is not enough to make normal cells into tumor cells. It is a current research focus that epigenetic modification which includes DNA methylation and histone acetylation has an important role in tumorigenesis. Gene promoter methylation is an early event in tumorigenesis and appears frequently in precancerous lesions of many tumors, so DNA methylation may be a molecular biology method in early diagnosis of cancer. Ras associated domain family 1A (RASSFIA) is a candidate tumor suppressor gene, the promoter methylation of which is related with the occurrence of many human epithelial tumors, such as lung cancer, breast cancer, gastric cancer, esophageal cancer, and so on. This study was designed to investigate the promoter methylation status and protein expression of RASSFIA gene in cervical carcinoma, in order to analyze the correlation between methylation and gene inactivation of RASSF1A, to explore the relationship between RASSF1A gene methylation and the occurrence of cervical carcinoma, and to provide a new theoretical basis of pathogenesis and early diagnosis of cervical carcinoma.Materials and Methods1 Study ObjectThe 60 fresh specimens were collected from these patients who were performed by gynecological surgery or outpatient biopsy in October 2008-December 2009 at the First Affiliated Hospital of Zhengzhou University. The clinical and pathological features of these patients were as follows:stageâ… 25 cases, stageâ…¡24 cases, stageâ…¢-â…£11 cases; squamous cell carcinoma 46 cases, adenocarcinoma 14 cases; grade G1-G2 39 cases, grade G3 21 cases; 7 cases with pelvic lymph nodes metastasis. The patients age was range from 25 to 67, and the mean age was 42 years. All specimens were pathologically diagnosed, and all patients did not receive preoperative radiotherapy and chemotherapy. In addition, we selected 15 cases of normal cervical tissues as control. Each specimen is divided into two. One was placed in the ice box immediately after cutting, and then transferred to -80℃refrigerator. The other was fixed with 4% formaldehyde solution, paraffin-embedded for immunohistochemistry staining.2 Experimental Methods2.1 Methylation-specific PCR (MSP)The basic principle of methylation-specific PCR is as follows:After genome DNA treatmented by bisulfite, all methylated cytosine (C) does not take place deamination reaction, but not-methylated cytosine (C) is translated into uracil (U) by deamination reaction. Based on this base changing, we can design non-methylated and methylation specific primers respectively, and separate methylated and unmethylated genes combined with PCR amplification.2.2 Immunohistochemical StainingExpression of RASSF1A protein is detected by avidin-biotin-horseradish peroxide enzyme complex method (ABC), and the working solution concentration of monoclonal mouse anti-human RASSF1A antibody is 1:500, which is purchased from eBioscience Corporation of U.S. 3 Statistical AnalysisAll datas are processed by SPSS 16.0 software package.x2 test, Fisher exact test and Spearman rank correlation analysis were used for statistical analysis. Significance level is a=0.05 orα'=0.017 corrected.Results1 Methylation status of RASSF1A gene and its relationship with clinicopathological characteristics of cervical carcinomaRASSF1A gene methylation was completely absent in 15 cases of normal cervical tissues, while in 60 cases of cervical carcinoma,21 cases took place RASSF1A gene methylation(35.0%), and the difference between these two group was significant (P <0.05). The methylation rate of RASSF1A gene was significantly higher in cervical squamous cell carcinoma than in adenocarcinoma (P<0.05). At stageâ… , stageâ…¡, stageâ…¢-â…£, the methylation rate of RASSF1A was gradually increased (16.0%â†'41.7%â†'63.6%, P<0.05), and the methylation rate of RASSF1A gene was significantly higher in stageâ…¢-â…£than in stage I (P<0.017). However, there was no evidence to demonstrate the relationship between RASSF1A hypermethylation and histological grade, lymph node metastasis (.P>0.05). 2 RASSF1A protein expression and its relationship with RASSF1A gene methylationAll normal cervical tissues expressed RASSF1A protein, while the expression rate of RASSF1A protein in cervical carcinoma was only 48.3%, and the difference was significant (P<0.05). The expression rate of RASSF1A protein was significantly lower in cervical squamous cell carcinoma than in adenocarcinoma (P<0.05). At stageâ… , stageâ…¡, stageâ…¢-â…£, the expression rate of RASSF1A protein was gradually decreased (72.0%â†'37.5%â†'-18.2%, P<0.05), and there were statistically significant between stageâ… and stageâ…¡, stageâ… and stageâ…¢-â…£(P<0.017). In addition, there was no evidence to demonstrate the relationship between RASSF1A protein expression and histological grade, lymph node metastasis (P>0.05).Moreover, Spearman rank correlation analysis showed that the methylation of RASSF1A was negatively correlated to its protein expression (r=-0.500, P<0.05).Conclusion1 There is an abnormal high methylation of RASSF1A gene promoter in cervical carcinoma, and this gene has two methylation forms, homozygous methylation and heterozygous methylation, which suggest that the methylation of RASSF1A gene has an relationship with occurrence of cervical carcinoma, and it is a frequent event in cervical carcinoma.2 The abnormal high methylation of RASSF1A gene promoter is very common in late clinical stage of cervical squamous cell carcinoma, which suggests that the methylation of RASSF1A gene may be associated with invasion and metastasis of cervical cancer cells.3 The promoter methylation of RASSF1A gene is an important mechanism that leads to RASSF1Ainactivation in cervical carcinoma. |
PDF Full Text Request