Difference in miRNA Expression in Functioning and Silent Corticotroph Pituitary Adenomas Indicates the Role of miRNA in the Regulation of Corticosteroid Receptors

Abstract

Corticotroph pituitary adenomas commonly cause Cushing's disease (CD), but some of them are clinically silent. The reason why they do not cause endocrinological symptoms remains unclear. We used data from small RNA sequencing in adenomas causing CD (n = 28) and silent ones (n = 20) to explore the role of miRNA in hormone secretion and clinical status of the tumors. By comparing miRNA profiles, we identified 19 miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. The analysis of their putative target genes indicates a role of miRNAs in regulation of the corticosteroid receptors expression. Adenomas causing CD have higher expression of hsa-miR-124-3p and hsa-miR-135-5p and lower expression of their target genes NR3C1 and NR3C2. The role of hsa-miR-124-3p in the regulation of NR3C1 was further validated in vitro using AtT-20/D16v-F2 cells. The cells transfected with miR-124-3p mimics showed lower levels of glucocorticoid receptor expression than control cells while the interaction between miR-124-3p and NR3C1 3' UTR was confirmed using luciferase reporter assay. The results indicate a relatively small difference in miRNA expression between clinically functioning and silent corticotroph pituitary adenomas. High expression of hsa-miR-124-3p in adenomas causing CD plays a role in the regulation of glucocorticoid receptor level and probably in reducing the effect of negative feedback mediated by corticosteroids.

Keywords: Cushing’s disease; NR3C1; glucocorticoid receptor; hsa-miR-124-3p; miRNA; neuroendocrine pituitary tumors; silent corticotroph adenoma.

Figure 1

MiRNA expression profiling in corticotroph adenomas. (A). Difference in miRNA expression between functioning and silent corticotroph adenomas. Volcano plot showing differentially expressed miRNAs. Significance and fold change thresholds are marked with dashed lines. (B). Heat map representing the expression of differentially expressed miRNAs and clustering the samples of adenomas causing Cushing’s disease (CD) and silent corticotroph adenomas (SCA). (C). The correlation between the expression levels of differentially expressed miRNAs and POMC expression or hormonal laboratory measurements in patients: morning plasma ACTH level, morning and midnight plasma cortisol levels and 24 h urinary free cortisol; * indicate p-value < 0.05; ** indicate p-value < 0.01; *** indicate p-value < 0.001

Figure 2

Gene set over-representation analysis of putative target genes of miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas.

Figure 3

The expression levels of NR3C1 and NR3C2 measured with qRT-PCR as well as hsa-miR-124-3p and hsa-miR-135b-5p measured with small RNA sequencing in tumor samples from CD patients and silent corticotroph adenomas; * indicate p-value < 0.05

Figure 4

Role of mir-124-3p in regulation of glucocorticoid receptor gene. (A). Putative hsa-mir-124-3p target sites in 3′UTR of NR3C1. (B). Reduced expression of Nr3c1 gene expression and glucocorticoid receptor (GR) protein level in AtT-20/D16v-F2 cells treated with hsa-miR-124-3p mimics. (C). Results of luciferase reporter gene assay, showing the interaction between Nr3c1 3′UTR site 2 and mir-124-3p; * indicate p-value < 0.05; ns—not significant.