Selective loss of MEG3 expression and intergenic differentially methylated region hypermethylation in the MEG3/DLK1 locus in human clinically nonfunctioning pituitary adenomas.
BACKGROUND
MEG3 is an imprinted gene encoding a novel noncoding RNA that suppresses tumor cell growth. Although highly expressed in the normal human pituitary, it is unknown which of the normal pituitary cell types and pituitary tumors express MEG3.


OBJECTIVE
Our objectives were 1) to investigate cell-type- and tumor-type-specific expression of MEG3 in the human pituitary and 2) to investigate whether methylation in the intergenic differentially methylated region (IG-DMR) at the DLK1/MEG3 locus is involved in the loss of MEG3 expression in tumors.


METHODS
RT-PCR, quantitative RT-PCR, Northern blot, and a combination of in situ hybridization and immunofluorescence were used to determine the cell-type- and tumor-type-specific MEG3 expression. Bisulfite treatment and PCR sequencing of genomic DNA were used to measure the CpG methylation status in the normal and tumor tissues. Five normal human pituitaries and 17 clinically nonfunctioning, 11 GH-secreting, seven prolactin-secreting, and six ACTH-secreting pituitary adenomas were used.


RESULTS
All normal human pituitary cell types express MEG3. However, loss of MEG3 expression occurs only in nonfunctioning pituitary adenomas of a gonadotroph origin. All other pituitary tumor phenotypes examined express MEG3. Hypermethylation of the IG-DMR at the DLK1/MEG3 locus is present in nonfunctioning pituitary adenomas.


CONCLUSIONS
MEG3 is the first human gene identified expressed in multiple normal human pituitary cell types with loss of expression specifically restricted to clinically nonfunctioning pituitary adenomas. The IG-DMR hypermethylation may be an additional mechanism for MEG3 gene silencing in such tumors.
Gejman
Roger
R
Neuroendocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Batista
Dalia L
DL
Zhong
Ying
Y
Zhou
Yunli
Y
Zhang
Xun
X
Swearingen
Brooke
B
Stratakis
Constantine A
CA
Hedley-Whyte
E Tessa
ET
Klibanski
Anne
A
D000236:Adenoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002135:Calcium-Binding Proteins; D002648:Child; D019175:DNA Methylation; D021901:DNA, Intergenic / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008875:Middle Aged; D010902:Pituitary Gland / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D040641:Quantitative Trait Loci / Q000235:genetics; D062085:RNA, Long Noncoding; D014018:Tissue Distribution
D002135:Calcium-Binding Proteins; C512941:DLK1 protein, human; D021901:DNA, Intergenic; D036341:Intercellular Signaling Peptides and Proteins; C498907:MEG3 non-coding RNA, human; D008565:Membrane Proteins; D011506:Proteins; D062085:RNA, Long Noncoding