Evaluation of methylation changes and the expression of RASSF2 gene in tumor tissue and tumor margins of breast cancer patients

Abstract

Today, cancer is known as the second cause of death after cardiovascular diseases (22%), its occurrence depends on factors such as age, genetic background and gender. According to published statistics, breast cancer is one of the most common cancers among women. According to the statistics of the World Health Organization, 1 out of every 8 to 10 women has breast cancer. RASSF2 is one of the 10 members of RASSF (Ras association domain family). Through interaction with Ras GTPase, these proteins regulate diverse cellular processes including membrane trafficking, apoptosis, migration and proliferation. RASSFs are classified as tumor suppressors based on their ability to inhibit the RAS protooncogene. Like other family members, RASSF2A can inhibit cell growth and induce apoptosis. Accordingly, loss of RASSF2A expression has been observed in various tumors and cancer cell lines, and such phenomena are often associated with RASSF2A promoter hypermethylation and poor prognosis for patients. In this study, the level of expression of this gene and its methylation in breast cancer patients and their relationship with the variables of age, smoking, family history, stage and metastasis have been investigated.

Methods: In this research, 75 tumor tissue samples and 75 tumor margin samples from cancer patients were examined. After extracting RNA (trizol) from tissues, cDNA synthesis was performed and it was used to evaluate changes in RASSF2 gene expression by Real Time PCR. Also, DNA was extracted from tissue samples and the methylation pattern of RASSF2 promoter regions was investigated using bisulfite treatment and MS-HRM (methylation sensitive high resolution melt analysis) technique. Statistical analysis was performed to investigate the changes in the expression and methylation of the RASSF2 gene and their effects on the clinical characteristics of the patients, and pvalue<0.05 was considered as a significant level. Using the statistical method of ROC curve analysis, the biomarker potential of RASSF2 gene expression and methylation was evaluated as a factor in breast cancer diagnosis.

Results: The obtained results have determined that there is a significant relationship between the expression of the gene in cancerous tissue and healthy tissue, so that the expression of the RASSF2 gene in breast cancer tissues is lower than in tumor peripheral tissues. Also, methylation in breast cancer tissues is clearly higher than healthy tissues.