Introduction: Ovarian cancer is the fifth leading cause of cancer related deaths. One of the main reasons for such high mortality is the lack of specific screening tests from physical and pelvic exams, and relatively little is known about the molecular events that lead to the development of this highly aggressive disease. The recent discovery of microRNAs (miRNA), a class of small non-coding RNAs that target other mRNAs and triggering translation repression and/or RNA degradation, has revealed the existence of a new level of gene expression regulation. Many studies involving various types of human cancers proved that miRNAs have a definitive role in tumorigenesis. Pyrosequencing is a real-time based sequencing technology that has been used widely for DNA methylation analysis and mutation detection. Materials and Methods: We developed 100 DNA gene promoter methylation assays using Pyrosequencing technology. So far we quantified DNA methylation in 19 candidate gene promoters and 16 mutation markers in two ovarian cancer cell lines (CAOV3 and SKOV3). The 19 candidate genes are known to be related to tumorigenesis. LINE element and Alu repeats are used as global methylation markers. The 16 mutation markers include KRAS, BRAF, and NRAS as well as several members of the cytochrome family of genes. Additionally, we have developed assays in the regions surrounding several miRNAs that have been shown to have altered regulation in ovarian tumors. These include, miR-200a,b and c, miR-141, miR-199a, miR-140, miR-145, miR-125b, and miR203. Expression analysis on the miRNAs was carried out using real-time PCR technology to determine the effects of the methylation state of the miRNA promoters on their expression Results: Among 19 promoter genes, RASSF1, p16 (CDKN2A), BRCA1, ESR1, and EFS are unmethylated, while TNFSF7 and KCNMA1 are fully methylated in both cell lines. Interestingly, SKOV3 showed higher methylation level than CAOV3 in some of genes. Line 1 and Alu has the methylation level of ~65%, and ~30% methylation, respectively. No KRAS, NRAS and Braf mutations were found in these 2 cell lines, however, there were several cell line-specific mutations identified using various SNP assays. Results of the miRNA methylation and expression analysis are currently being compiled. Conclusions: Our preliminary results demonstrate that Pyrosequencing allows the analysis of a combination of genetic and epigenetic markers in a single analysis and confirm that certain genes are highly methylated in their promoter region. We were able to analyze over 20 methylation markers, including novel miRNA assays, and over 20 mutational markers in both cell lines. In addition, expression analysis was correlated between the miRNA methylation assays and expression levels using real-time PCR. In combination, these technologies are valuable tools in the analysis of epigenetic events in cancerous cells.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3359.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009