Metilovanje promotora i ekspresija estrogenskog receptora beta (ERß) u invazivnim karcinomima dojke

Abstract

Invazivni karcinom dojke je najčešći kancer kod žena. Pored genetičkih i epigenetički faktori imaju važnu ulogu u njegovoj inicijaciji i progresiji. Cilj ove studije bio je ispitati da li metilacija promotora ON, gena za ERβ protein (ESR2) utiče na njegovu ekspresiju (na nivou iRNK i proteina) i utvrditi povezanost metilacionog indeksa promotora ON sa nivoima ERβ1-iRNK i ERβ1 proteina, kao i sa kliničkim i patohistološkim parametrima. U ovoj studiji analiziran je 131 arhivski uzorak kancera dojke. Pomoću posebno dizajniranog PCR testa iz dva koraka, amplifikovan je i kvantifikovan specifični region promotora ON ESR2 gena. Za kvantifikaciju ERβ1-iRNK korišćena je metoda kvantitativnog RT-PCR u realnom vremenu. Metoda „Western Blot“ je korišćena za relativnu kvantifikaciju ERβ1 proteinske izoforme. Dobijeni podaci, metilacioni indeks promotora ON, nivoi iRNK i ERβ1 proteina, analizirani su i korelisani sa kliničkopatološkim parametrima neparametrijskim statističkim testovima. Nađeno je da je metilacioni indeks promotora ON ERβ gena značajno veći u grupi pacijentkinja sa pozitivnim, u odnosu na grupu sa negativnim statusom aksilarnih limfnih čvorova. Takođe, pronađena je značajna pozitivna korelacija metilacionog indeksa promotora ON ERβ gena sa nivoom estrogenskog receptora α. Na osnovu dobijenih rezultata pokazano je da metilacioni indeks promotora ON, ERβ gena može biti pouzdaniji marker prognoze kancera dojke nego ekspresija ERβ1-iRNK ili proteina ERβ1.

Breast cancer is the most common invasive cancer in women. Beside genetic, epigenetic factors influence its initiation and progression. The purpose of this study was to test whether methylation of the promoter ON of the gene for ERβ protein (ESR2) influences its expression (on mRNA and protein level) and to correlate the methylation index of the ON promoter, ERβ1-mRNA and ERβ1 protein levels with clinicopathological parameters. In this study, 131 archival samples of breast cancer tissue were used. Custom designed two step PCR method, was done for amplification and relative quantification of the specific region of the ON promoter of the ESR2 gene. Measuring of ERβ1-mRNA was done by quantitative real time RT-PCR, and relative quantification of ERβ1 protein isoform was done by Western blot. Statistical analysis was performed and results for methylation index of ON promoter, mRNA and ERβ1 protein levels were correlated with clinicopathological parameters. The methylation index of ERβ-ON promoter was significantly higher in a group of patients with positive axillary lymph node status, than in a group with negative one. A significant positive correlation between methylation index of ON promoter and ERα protein levels was obtained. It can be concluded that methylation index of the ERβ-ON promoter could be more confident parameter for prediction and/or prognosis of breast cancer, than measuring of ERβ1-mRNA or ERβ1 protein levels.