ECE2022 Eposter Presentations Pituitary and Neuroendocrinology (211 abstracts)
Circulating plasma microRNA in patients with ACTH-dependent cushing’s syndrome.
Anastasia Malygina 1 , Zhanna Belaya 1 , Alexander Solodovnikov 2 , Alexey Nikitin 3 , Philipp Koshkin 4 , Ivan Sitkin 1 , Michael Pikunov 5 , Patimat Khandaeva 1 , Alexander Lutsenko 1 , Diana Trukhina 1 , Anastasia Lapshina 1 , Andrey Grigoriev 1 , Galina Melnichenko 1 & Ivan Dedov 1
1The National Medical Research Centre for Endocrinology, neuroendocrinology and bone disease, Moscow, Russian Federation; 2Ural State Medical University, Department of Preventive and Family Medicine, Moscow, Russian Federation; 3Pulmonology Scientific Research Institute under FMBA of Russia, Moscow, Russian Federation; 4Laboratory of Molecular Pathology, Center of Medical Genetics (Genomed), Moscow, Russian Federation; 5National Medical Research Center of Surgery Named After A.V. Vishnevsky, Moscow, Russian Federation
Introduction: Recent studies have shown that microRNA could serve as biomarkers in various types of cancer and other diseases.
Aim: To reveal microRNA that differ in patients with Cushing’s diseases (CD) and Ectopic ACTH-syndrome (EAS) to form a specific panel for differential diagnosis of ACTH-dependent Cushing’s syndrome (CS).
Materials and Methods: Plasma samples from both sinuses and cubital vein were drained during inferior petrosal sinus sampling and stored at -80 C. MiRNA isolation from plasma samples was carried out by an Rneasy Plasma/Serum Kit (Qiagen, Germany) on the automatic QIAcube station. MiR expression was then analyzed by sequencing on Illumina NextSeq 500 (Illumina, USA). The libraries were prepared by the QIAseq miRNA Library Kit. Sequencing was performed on a total of 36 samples. Data analysis and interpretation was conducted on Qiagen GeneGlobe Data Analysis were Center. qRT-PCR was performed using a TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher, Scientific) and TaqMan® Advanced miR Assays (Thermo Fisher Scientific), in a 96-well format on the StepOnePlus instrument (Applied Biosystems). Data analyses were performed using SDS software (version 2.3, Applied Biosystems), to obtain cycle treshold (Ct) data. We used value Ct <35 as a cutoff of detection. All samples were normalized to spike-in control, cel-miR-39-3p.
Results: Among 36 enrolled patients (mean age 47,5 years (minimum 23, maximum 69 years; M:7, F:29) 24 subjects were confirmed as CD and 12 as EAS. There were 1167 miRNA differently detected (P<0,05) in inferior petrosal sinus samples of patients with CD vs EAS. These miRNAs were divided 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. 108 microRNA were included. For the verification phase 10 microRNA were chosen (miR-383-3p, miR-4290, miR-6717-5p, miR-1203, miR-1229-3p, miR-639, miR-302c-3p, miR-7g-5p, miR-145-5p, miR-16-5p) according to the discovery phase results and data from the previous pilot study. We enrolled 82 patients (mean age 44,5 years (minimum 19, maximum 70 years; M:18, F:64) for validation phase of the study. Among them 64 were confirmed as CD, 18 as EAS. RT-qPCR showed, that four microRNA differ between patients with CD and EAS: miR-383-3p (padjusted=0,003), miR-302c-3p (padjusted=0,02), miR-4290 (padjusted=0,02), miR-6717-5p (padjusted=0,02).
Conclusion: miR-383-3p, miR-302c-3p, miR-4290, miR-6717-5p differed between patients with CD and EAS in peripheral plasma samples as measured by both NGS and qRT-PCR and could form a specific panel for differential diagnosis of ACTH-dependent CS.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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