Clinical & Medical Biochemistry

Abstract

Molecular Analysis of RASSF1 Gene Methylation and mRNA Expression in Sporadic Breast Cancer

Maria Lagiou, Nikoleta Poumpouridou, Nikolaos Goutas, Dimitrios Vlachodimitropoulos, Aggeliki Pappa, Evi Lianidou, Triantafyllos Liloglou and Christos Kroupis

Introduction: The aim of this study was to validate innovative and reliable methods for studying DNA methylation of the first promoter of RASSF1 gene (where its primary transcript RASSF1A in breast tissue is transcribed from), its mRNA expression and their evaluation in sporadic breast cancer. Materials and methods: DNA and RNA from 81 frozen breast cancer tissues with known histopathological data as well as 4 normal breast tissues were analyzed. DNA methylation levels were assessed by Pyrosequencing, analyzing 9 CpG dinucleotides in the CpG island of the first RASSF1 promoter. For mRNA expression, a real-time RT-qPCR method was used and validated with synthetic standards, with the SYBR Green PCR kit and a suitable set of standardized primers (Qiagen) for all RASSF1 transcript variants (except G). For relative quantification of RASSF1 expression, the 2-ΔCt method was used with beta2-microglobulin as a reference gene. Results: 59 samples were characterized as RASSF1 normally-methylated (72.8%), while 22 samples as hypermethylated (27.2%). Also, 40 samples were classified as RASSF1 mRNA over-expressing (49.4%), while 41 (50.6%) as sub-expressing. No inverse correlation was found between methylation and RASSF1 expression (p=0.207). Logistic regression showed that the probability of lymph node infiltration was increased by the presence of negative ER receptors (p=0.008, OR 0.09, CI 0.14-0.52), the percentage of methylation (p=0.006, OR 7.96, CI 1.82-34.86) and the level of RASSF1 expression (p=0.047, OR 3.94, CI 1.02-15.29). Survival analysis showed that there is a marginal statistically significant correlation between metastasis and mRNA RASSF1 over-expression (log rank test, p=0.040). Conclusions: The evaluated assays appear to provide potentially useful information for the clinical management of breast carcinomas. A similar reliable assay for the methylation of the second RASSF1 promoter should be evaluated in the future. Regarding the expression study, a new design strategy with probes specific for all different RASSF1 transcripts, would provide additional power in the study and potentiate the use of new RASSF1 biomarkers in the clinical evaluation of sporadic breast cancer.