ZIC1 is silenced and has tumor suppressor function in malignant pleural mesothelioma.
BACKGROUND
Epigenetic inactivation of tumor suppressor genes is involved in the development of malignant pleural mesothelioma (MPM). ZIC1, a potential tumor suppressor gene involved in regulating cell growth and apoptosis, was investigated in MPM cell lines and tumors.


METHODS
ZIC1 expression and promoter methylation were evaluated in MPM cell lines and tumor samples by quantitative polymerase chain reaction (PCR), Combined Bisulfite Restriction Analysis, and methylation-specific PCR. ZIC1 was reexpressed in cell lines and functional effects were assessed. miRNA expression was quantified by microarray and reverse transcription quantitative PCR. ZIC1 knockdown and miRNA inhibitors were used to study the relationship between ZIC1 and miRNA expression and confirmed by chromatin immunoprecipitation PCR.


RESULTS
ZIC1 expression was low in MPM cells, and was correlated with ZIC1 promoter methylation and reversed upon decitabine treatment. ZIC1 reexpression inhibited proliferation and invasion in MPM cells whereas knockdown enhanced the growth of MeT-5A. In MPM tumor samples ZIC1 expression was either low or undetectable, with promoter methylation observed in 16 of 24 cases. The overexpression of miR-23a and miR-27a was reduced by ZIC1 reexpression, with inhibitors of miR-23a or miR-27a reducing colony formation. miR-23a overexpression was also associated with shorter survival of MPM patients.


CONCLUSIONS
ZIC1 is down-regulated in MPM through promoter methylation and acts as a tumor suppressor through down-regulation of its direct targets miR-23a and miR-27a.
*Asbestos Diseases Research Institute, University of Sydney, Sydney, Australia; †Department of Anatomical Pathology, Flinders Medical Centre, Adelaide, Australia; ‡Cardiothoracic Surgical Unit, Royal Prince Alfred Hospital; The Baird Institute, Sydney Medical School, University of Sydney, Sydney, Australia; and §Biomedical Research Center, Sir Runrun Shaw Hospital, Zhejiang University, Hangzhou, China.
Yuen Yee
YY
Cheng
Michaela B
MB
Kirschner
Ngan Ching
NC
Cheng
Sumedha
S
Gattani
Sonja
S
Klebe
J James B
JJ
Edelman
Michael P
MP
Vallely
Brian C
BC
McCaughan
Hong Chuan
HC
Jin
Nico
N
van Zandwijk
Glen
G
Reid
D000328:Adult; D000368:Aged; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D005260:Female; D005455:Fluorescent Antibody Technique; D005500:Follow-Up Studies; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007124:Immunoenzyme Techniques; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D008297:Male; D008654:Mesothelioma / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D000086002:Mesothelioma, Malignant; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009367:Neoplasm Staging; D020411:Oligonucleotide Array Sequence Analysis; D010997:Pleural Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology; D011379:Prognosis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; C492123:ZIC1 protein, human