PO-380 Epigenetically regulated MAF is a new potential tumour suppressor gene in LSCC

Abstract

IntroductionLaryngeal cancer (LSCC) is one of the most common tumours among head and neck squamous cell carcinomas. Recently, analysis of the meaning of epigenetic alterations in LSCC development has gained momentum. In this field we have shown recurrent overexpression of the miR-1290 in LSCC. Therefore, in this study we aim at clarifying the oncomiR functionality of miR-1290 by analysing its interaction with the transcriptional modulator MAF – a putative suppressor gene.Material and methodsThe identification of miR-1290 overexpression in LSCC was based on global miRNA expression profiles (Agilent) of 16 LSCC cell lines and 3 non-tumour controls which were validated on 50 tumour samples by real-time qPCR (RT-qPCR) with LNA modified primers. Target genes of miR-1290 were selected by in silico analysis using miRDB (TS >90) and miRWALK (gene listed in at least 2 in 4 databases). The influence of miR-1290 on its putative target gene MAF was analysed by transient transfection of 2 LSCC cell lines using miRNA inhibitor and successive expression analysis of MAF in the transfected cells by RT-qPCR and Western blot. To confirm the downregulation of MAF in primary LSCC we conducted RT-qPCR on 22 tumour samples and 5 non-tumour controls. In addition, the nuclear MAF protein abundance was evaluated by immunohistochemistry on tissue microarray containing 128 LSCC tumour samples and 19 tumour free resection margins.Results and discussionsBased on the in sillico analysis MAF has been selected as a the putative miR-1290 target gene with high suppressor potential. Importantly, MAF is a transcription factor, involved in regulation of apoptosis and TP53 expression, processes deregulated in LSCC. Functional analyses, showed that inhibition of miR-1290 resulted in 3.47 fold increase in MAF mRNA expression and 50% increase of MAF protein abundance. Moreover, by using RT-qPCR we have shown recurrent MAF downregulation in LSCC tumour samples compared to non-tumour controls. In line with these findings we have indicated by immunohistochemical staining that 74/128 LSCC tumour samples were characterised by lack of nuclear expression of MAF in opposite to nuclear expression observed in 19/19 margins.ConclusionOur results demonstrate MAF as a new potential tumour suppressor gene epigenetically downregulated in LSCC by the overexpression of miR-1290. Further studies are required for fully understanding the role of MAF in LSCC pathogenesis.