Endocrine Abstracts

Introduction: ACTH-secreting pituitary adenoma tissue differs from normal pituitary gland tissue in microRNA (miR) expression. MiRs are secreted into circulation and can be detected as potential biomarkers

Aim: To evaluate the difference in miR levels in plasma samples drained from inferior petrosal sinuses in patients with Cushing’s disease (CD) and ectopic-ACTH syndrome (EAS).

Materials and methods: we enrolled 24 patients with ACTH-dependent Cushing’s syndrome (CS) requiring bilateral inferior petrosal sinus sampling (BIPSS). BIPSS was performed through a percutaneous bilateral approach. Once catheters were properly placed, blood samples were withdrawn simultaneously from each petrosal sinus and a peripheral vein. In addition to ACTH, prolactin measurements were used as additional proof for correct catheter placement. Plasma samples from both sinuses were stored at –80°C. MiRNA isolation from plasma was carried out by an Rneasy Plasma/Serum Kit (Qiagen, Germany) on the automatic QIAcube station according to the manufacturer protocol. To prevent degradation, we added 1 unit of RiboLock Rnase Inhibitor (Thermo Fisher Scientific, USA) per 1 µl of RNA solution. The concentration of total RNA in the aqueous solution was evaluated on a NanoVue Plus spectrophotometer (GE Healthcare, USA).MiR expression was then analyzed by sequencing on Illumina NextSeq 500 (Illumina, USA). The libraries were prepared by the QIAseq miRNA Library Kit following the manufacturer standard protocols. Sequencing was performed on a total of 24 samples. Data analysis and interpretation was conducted on Qiagen GeneGlobe Data Analysis Center.

Results: Among 24 enrolled patients (mean age 48 years (minimum 23, maximum 69); M:6, F:18) 12 subjects were confirmed as CD and 12 as EAS. 108 miRNA were differently detected (P <0,05) in inferior petrosal sinus samples of patients with CD vs EAS. We divided these miRNAs into 3 groups based on the significance of the results. The first group consisted of samples with the highest levels of detected miR in both groups. Four miRNAs were included: hsa-miR-1203 was downregulated in CD vs EAS–36.74 (P = 0,013), and three other were upregulated in CD vs EAS: hsa-miR-383-3p 46.36 (P = 0,01), hsa-miR-4290 6.84 (P = 0,036), hsa-miR-6717-5p 4.49 (P = 0,031). The vast majority of differently expressed miRNAs are involved in oncogenesis signaling pathways.

Conclusion: Plasma miR levels differ in inferior petrosal samples taken from patients with CD vs EAS. These miRs need to be validated by different methods and in peripheral plasma samples in order to be used as potentially non-invasive biomarkers to differentiate ACTH-dependent CS.