Abstract
IntroductionPatients with germline AIP mutations or low AIP protein expression have large, invasive somatotroph adenomas and poor response to somatostatin analogues (SSA).MethodsTo study the mechanism of low AIP protein expression 31 sporadic somatotropinomas with low (n = 13) or high (n = 18) AIP protein expression were analyzed for expression of AIP messenger RNA (mRNA) and 11 microRNAs (miRNAs) predicted to bind the 3’UTR of AIP. Luciferase reporter assays of wild-type and deletion constructs of AIP-3’UTR were used to study the effect of the selected miRNAs in GH3 cells. Endogenous AIP protein and mRNA levels were measured after miRNA over- and underexpression in HEK293 and GH3 cells.ResultsNo significant difference was observed in AIP mRNA expression between tumors with low or high AIP protein expression suggesting post-transcriptional regulation. miR-34a was highly expressed in low AIP protein samples compared high AIP protein adenomas and miR-34a levels were inversely correlated with response to SSA therapy. miR-34a inhibited the luciferase-AIP-3’UTR construct, suggesting that miR-34a binds to AIP-3’UTR. Deletion mutants of the 3 different predicted binding sites in AIP-3’UTR identified the c.*6–30 site to be involved in miR-34a’s activity. miR-34a overexpression in HEK293 and GH3 cells resulted in inhibition of endogenous AIP protein expression.ConclusionLow AIP protein expression is associated with high miR-34a expression. miR-34a can down-regulate AIP-protein but not RNA expression in vitro. miR-34a is a negative regulator of AIP-protein expression and could be responsible for the low AIP expression observed in somatotropinomas with an invasive phenotype and resistance to SSA.
Highlights
Patients with germline Aryl Hydrocarbon Receptor Interacting Protein (AIP) mutations or low AIP protein expression have large, invasive somatotroph adenomas and poor response to somatostatin analogues (SSA).Received: October 4, 2014Accepted: December 19, 2014Published: February 6, 2015
No significant difference was observed in AIP messenger ribonucleic acid (RNA) (mRNA) expression between tumors with low or high AIP protein expression suggesting post-transcriptional regulation. miR-34a was highly expressed in low AIP protein samples compared high AIP protein adenomas and miR-34a levels were inversely correlated with response to SSA therapy. miR-34a inhibited the luciferase-AIP-3’UTR construct, suggesting that miR-34a binds to AIP-3’UTR
Low AIP protein expression is associated with high miR-34a expression. miR-34a can down-regulate AIP-protein but not RNA expression in vitro. miR-34a is a negative regulator of AIP-protein expression and could be responsible for the low AIP expression observed in somatotropinomas with an invasive phenotype and resistance to SSA
Summary
Luciferase reporter assays of wild-type and deletion constructs of AIP-3’UTR were used to study the effect of the selected miRNAs in GH3 cells. Thirty-four consecutive patients with acromegaly who had previously had pituitary surgery and had tissue available (paraffin block and fresh frozen tumor sample) were included in the study. Exclusion criteria included previous known AIP mutations, a family history of pituitary adenoma, presence of features or family history of Carney complex or multiple endocrine neoplasia type 1 or 4 and preoperative therapy with SSA [as treatment may increase AIP expression [17]]. GH-secreting pituitary tumor samples were obtained during transsphenoidal surgery: part of the sample was processed for routine histopathological and immunohistochemical studies (including anterior pituitary hormones), and part was snap-frozen and stored at -70°C for molecular biology studies. Five normal human pituitaries were obtained within 10 hours from the time of death at autopsies of subjects who had died from natural causes without previous evidence of any endocrine disease or pituitary abnormality
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