MicroRNA signature in massive macronodular adrenocortical disease and implications for adrenocortical tumourigenesis

Abstract

Purpose: Massive macronodular adrenocortical disease (MMAD) may be caused by aberrant microRNA expression. To determine the microRNA profile in MMAD and identify putative microRNA-gene target pairs involved in adrenal tumourigenesis.

Experimental design: We performed microRNA microarray analysis in 10 patients with ACTH-independent Cushing syndrome caused by MMAD (ages 39-60 years) and four normal adrenal cortex samples were used as controls. Microarray data were validated by real-time polymerase chain reaction (qRT-PCR). Identification of potential microRNA-gene target pairs implicated in MMAD pathogenesis has been performed by integrating our microRNA data with previously obtained cDNA microarray data. Experimental validation of specific microRNA gene targets was performed by transfection experiments and luciferase assay.

Results: A total of 37 microRNAs were differentially expressed between MMAD and normal tissues; 16 microRNAs were down-regulated, including miR-200b and miR-203, whereas 21 microRNAs were up-regulated, miR-210 and miR-484 among them. Comparison of microRNA data with different clinicopathological parameters revealed miR-130a and miR-382 as putative diagnostic MMAD markers. Interestingly, we detected miR-200b targeting directly Matrin 3 (MATR3) expression in an adrenocortical cancer cell line (H295R).

Conclusions: MicroRNAs appear to have distinct regulatory effects in MMAD, including an association with clinical presentation and severity of the disease, expressed by the degree of hypercortisolism. This is the first investigation of microRNAs in MMAD, a disease with complex pathogenesis; the data indicate that specific microRNAs such as miR-200b may play a significant role in MMAD formation and/or progression.

Figure 1. Heat map representation of the expression pattern for 365 microRNAs in 10 MMAD samples and 4 normal adrenals

A color code is used for the level of microRNA expression: blue: lower microRNA expression, red: higher microRNA expression. The cut-off for differentially expressed microRNAs was 1.5-fold (up- or down-regulated). MMAD tissues are shown as M1-10; C1 is whole normal adrenal tissue (Ambion); C2 is whole normal adrenal tissue (Biochain); C3 is a pool of adrenal RNA from 5 normal subjects (Biochain) and C4 is normal adrenal cortex from a patient.

Figure 2. Real-time PCR analysis of differentially expressed microRNAs between MMAD and normal adrenal tissues

In order to validate microRNA microarray data, we performed microRNA SYBR Green Real-time PCR analysis. The results were consistent with the microarray data, showing that 16 microRNAs were down-regulated while 21 microRNAs were up-regulated in MMAD tissues relative to normal tissues. Statistical analysis was performed by using the SPSS statistics software package (SPSS). All results were expressed as mean ± SD, and P < 0.05 was used for significance. The cut-off for differentially expressed microRNAs was 1.5-fold (up- or down-regulated). MicroRNA expression data were normalized to U6 expression levels (loading control).

Figure 3. Correlation of MicroRNAs with clinical data

A, miR-382 and B, miR-130a expression have a positive correlation with midnight cortisol levels in MMAD patients. Correlation between microRNAs and midnight cortisol levels (nmol/L) was calculated with correlation coefficient.

Figure 4. Strategy for identifying microRNA gene targets in MMAD

We used TargetScan prediction algorithms in order to identify potential gene targets for the differentially expressed microRNAs between MMADs and normal adrenal tissues. From the predicted gene targets we selected only those that were differentially expressed in MMADs according to the cDNA microarray data (Bourdeau I et al, Oncogene, 2004). The last step was to select only the inverse correlated microRNA - gene target pairs, because microRNAs are negative regulators of gene expression.

Figure 5. MicroRNA-target gene pairs sequences

Identification of the specific sequences in the 3’UTR of microRNA gene target genes according to TargetScan Program Analysis.

Figure 6. miR-200b regulates directly MATR3 expression

A, qRT-PCR analysis of MATR3 expression levels in an adrenocortical cancer cell line (H295R cell line). Transfection of miR-200b in the H295R cell line (50nM, 100nM) reduced MATR3 expression (>50% and 60% suppression of mRNA expression respectively). B, Luciferase assay after transfection of miR-200b (100 nM) together with luciferase vector harboring the 3’UTR of MATR3 gene in HEK293 cells. More than 45% of MATR3 3’UTR luciferase activity was inhibited by miR-200b. The data are presented as mean ± SD of three independent experiments.