Methylation-associated Silencing of microRNA-126 and its Host Gene EGFL7 in Malignant Pleural Mesothelioma.
OBJECTIVE
We recently reported that miR-126 is down-regulated in malignant pleural mesothelioma (MPM) and can be combined into a 4-microRNA-classifier that can accurately diagnose MPM with high sensitivity and specificity. Herein we analyzed the epigenetic regulation of miR-126 and its host gene EGF-like domain, multiple 7 (EGFL7).


METHODS
Resected formalin-fixed paraffin-embedded MPM tissues from 29 patients, 14 patient-matched non-neoplastic pleura (NNP) specimens, 5 MPM diagnostic biopsies (DB), and 5 samples of pneumothorax-induced benign reactive mesothelial proliferation (PTHX) were analyzed. miR-126 and EGFL7 mRNA were quantified by RT-qPCR. CpG-islands' methylation in the EGFL7 promoter was analyzed using methylation-specific PCR and in the MIR126-containing intron 7 was quantified by pyrosequencing.


RESULTS
Relative to NNP, EGFL7 was under-expressed more than 4-fold in MPM (p<0.001). EGFL7 mRNA and miR-126 levels correlated in MPM (p<0.01) and NNP (p<0.001). The EGFL7 promoter region was hypermethylated in 69% of MPM and 80% of DB samples, but not in NNP and PTHX samples. EGFL7 promoter hypermethylation was associated with epithelioid histology (p<0.05) and reduced patient-survival (p<0.05).


CONCLUSIONS
In MPM, DNA-hypermethylation down-regulates miR-126 and its host gene EGFL7, therefore is a poor prognostic factor, and may represent a future therapeutic target for de-methylating strategies re-establishing EGFL7 and miR-126 expression.
Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
Morten
M
Andersen
Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
Davide
D
Trapani
Department of Thoracic Surgery, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
Jesper
J
Ravn
Department of Oncology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
Jens Benn
JB
Sørensen
Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark.
Claus Bøgelund
CB
Andersen
Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark eric.santoni-rugiu.02@regionh.dk morten.grauslund@regionh.dk.
Morten
M
Grauslund
Department of Pathology, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark eric.santoni-rugiu.02@regionh.dk morten.grauslund@regionh.dk.
Eric
E
Santoni-Rugiu
D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics*; D002135:Calcium-Binding Proteins; D016022:Case-Control Studies; D019175:DNA Methylation; D066255:EGF Family of Proteins; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism*; D044127:Epigenesis, Genetic; D005260:Female; D005500:Follow-Up Studies; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008654:Mesothelioma / Q000235:genetics* / Q000473:pathology; D000086002:Mesothelioma, Malignant; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics; D008875:Middle Aged; D009367:Neoplasm Staging; D010997:Pleural Neoplasms / Q000235:genetics* / Q000473:pathology; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
D014408:Biomarkers, Tumor; D002135:Calcium-Binding Proteins; D066255:EGF Family of Proteins; C527877:EGFL7 protein, human; D016228:Endothelial Growth Factors; C527111:MIRN126 microRNA, human; D035683:MicroRNAs
DNA-methylation; EGFL7; Malignant pleural mesothelioma; biomarker; miR-126; prognosis