Quantitative PCR from human genomic DNA: The determination of gene copy numbers for congenital adrenal hyperplasia and RCCX copy number variation.

Quantitative PCR (qPCR) is used for the determination of gene copy number (GCN). GCNs contribute to human disorders, and characterize copy number variation (CNV). The single laboratory method validations of duplex qPCR assays with hydrolysis probes on CYP21A1P and CYP21A2 genes, residing a CNV (RCCX CNV) and related to congenital adrenal hyperplasia, were performed using 46 human genomic DNA samples. We also performed the verifications on 5 qPCR assays for the genetic elements of RCCX CNV; C4A, C4B, CNV breakpoint, HERV-K(C4) CNV deletion and insertion alleles. Precision of each qPCR assay was under 1.01 CV%. Accuracy (relative error) ranged from 4.96±4.08% to 9.91±8.93%. Accuracy was not tightly linked to precision, but was significantly correlated with the efficiency of normalization using the RPPH1 internal reference gene (Spearman's ρ: 0.793-0.940, p>0.0001), ambiguity (ρ = 0.671, p = 0.029) and misclassification (ρ = 0.769, p = 0.009). A strong genomic matrix effect was observed, and target-singleplex (one target gene in one assay) qPCR was able to appropriately differentiate 2 GCN from 3 GCN at best. The analysis of all GCNs from the 7 qPCR assays using a multiplex approach increased the resolution of differentiation, and produced 98% of GCNs unambiguously, and all of which were in 100% concordance with GCNs measured by Southern blot, MLPA and aCGH. We conclude that the use of an internal (in one assay with the target gene) reference gene, the use of allele-specific primers or probes, and the multiplex approach (in one assay or different assays) are crucial for GCN determination using qPCR or other methods.

Circulating miRNA Increases the Diagnostic Accuracy of Chromogranin A in Metastatic Pancreatic Neuroendocrine Tumors.

Chromogranin A (CgA) is the most widely accepted biomarker for neuroendocrine tumors (NET) but its diagnostic accuracy is dependent on tumor type and the use of proton-pump inhibitors (PPI). We investigated the diagnostic value of circulating miRNAs along with CgA in pancreatic neuroendocrine tumors (pNET). 74 serum samples from patients with pNET (n = 25, nonfunctioning), pheochromocytoma/paraganglioma (PPGL, n = 20), healthy individuals with normal CgA (n = 29) including 10 samples from 5 healthy individuals with and without current PPI treatment were collected. MiRNA expression profiles were determined using next-generation sequencing, followed by validation with individual TaqMan assays. A global downregulation of miRNAs was observed in patients with NET compared to controls. MiRNA expression of 33 miRNAs was able to discriminate tumor samples from controls. No miRNA alone could be considered as an applicable biomarker for pNET or PPGL. However, using a logistic model, the combination of a set of miRNAs increased the discriminatory role of CgA irrespective of PPI treatment. In pNET patients with normal CgA level our regression model yielded high (89.4%) diagnostic accuracy (AUC: 0.904, sensitivity: 66.6%, specificity: 96.5%). A set of miRNAs increased the diagnostic utility of CgA in pNET even in patients with low CgA.

Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chromaffin cell line), Hela (human cervix epithelial cell line), and H295R (human adrenocortical cell line) cells, we demonstrated that chromaffin cells were not affected negatively by the inhibition of SDH either by siRNA directed against SDHB or treatment with SDH inhibitors (itaconate and atpenin A5). Cell viability and intracellular metabolite measurements pointed to the cell line specific consequences of SDH impairment and to the importance of glutamate metabolism in chromaffin cells. A significant increase in glutaminase-1 (GLS-1) expression after SDH impairment was observed in PC12 cells. GLS-1 inhibitor BPTES was capable of significantly decreasing proliferation of SDH impaired PC12 cells. Glutaminase-1 and SDHB expressions were tested in 35 Pheo/PGL tumor tissues. Expression of GLS1 was higher in the SDHB low expressed group compared to SDHB high expressed tumors. Our data suggest that the SDH-associated malignant potential of Pheo/PGL is strongly dependent on GLS-1 expression and glutaminases may be novel targets for therapy.

Circulating miRNA Expression Profiling in Primary Aldosteronism.

Objective: Primary aldosteronism is a major cause of secondary hypertension. Its two principal forms are bilateral adrenal hyperplasia (BAH) and aldosterone-producing adenoma (APA) whose differentiation is clinically pivotal. There is a major clinical need for a reliable and easily accessible diagnostic biomarker for case identification and subtyping. Circulating microRNAs were shown to be useful as minimally invasive diagnostic markers. Our aim was to determine and compare the circulating microRNA expression profiles of adenoma and hyperplasia plasma samples, and to evaluate their applicability as minimally invasive markers. Methods: One hundred and twenty-three samples from primary aldosteronism patients were included. Next-generation sequencing was performed on 30 EDTA-anticoagulated plasma samples (discovery cohort). Significantly differently expressed miRNAs were validated by real-time reverse transcription-qPCR in an independent validation cohort (93 samples). Results: We have found relative overexpression of miR-30e-5p, miR-30d-5p, miR-223-3p, and miR-7-5p in hyperplasia compared to adenoma by next-generation sequencing. Validation by qRT-PCR confirmed significant overexpression of hsa-miR-30e-5p, hsa-miR-30d-5p, and hsa-miR-7-5p in hyperplasia samples. Regarding the microRNA expressional variations, adenoma is more heterogeneous at the miRNA level compared to hyperplasia. Conclusion: Three microRNAs were significantly overexpressed in hyperplasia samples compared to adenoma samples, but their sensitivity and specificity values are not good enough for introduction to clinical practice.

Comprehensive Analysis of Circulating miRNAs in the Plasma of Patients With Pituitary Adenomas.

BACKGROUND: Circulating miRNAs in pituitary adenomas would improve patient care, especially as minimally invasive biomarkers of tumor recurrence and progression in nonfunctioning adenoma cases. AIM: Our aim was to investigate plasma miRNA profiles in patients with pituitary adenomas. MATERIALS AND METHODS: A total of 149 plasma and extracellular vesicle (preoperative, early postoperative, and late postoperative) samples were collected from 45 patients with pituitary adenomas. Adenomas were characterized on the basis of anterior pituitary hormones and transcription factors by immunostaining. miRNA next-generation sequencing was performed on 36 samples (discovery set). Individual TaqMan assays were used for validation on an extended sample set. Pituitary adenoma tissue miRNAs were evaluated by TaqMan array and data in the literature. RESULTS: Global downregulation of miRNA expression was observed in plasma samples of pituitary adenomas compared with normal samples. Expression of 29 miRNAs and isomiR variants were able to distinguish preoperative plasma samples from normal controls. miRNAs with altered expression in both plasma and different adenoma tissues were identified. Three, seven, and 66 miRNAs expressed differentially between preoperative and postoperative plasma samples in GH-secreting, FSH/LH+, and hormone-immunonegative groups, respectively. miR‒143-3p was downregulated in late postoperative but not in early postoperative plasma samples compared with preoperative ones exclusively in FSH/LH+ adenomas. The plasma level of miR‒143-3p discriminated these samples with 81.8% sensitivity and 72.3% specificity (area under the curve = 0.79; P = 0.02). CONCLUSIONS: Differentially expressed miRNAs in pituitary adenoma tissues have low abundance in plasma, minimizing their role as biomarkers. Plasma miR‒143-3p level decreased in patients with FSH/LH+ adenomas, indicating successful surgery, but its application for evaluating tumor recurrence needs further investigation.

Differentially Expressed miRNAs Influence Metabolic Processes in Pituitary Oncocytoma.

Spindle cell oncocytomas (SCO) of the pituitary are rare tumors accounting for 0.1-0.4% of all sellar tumors. Due to their rarity, little information is available regarding their pathogenesis. Our aim was to investigate miRNA expression profile of pituitary oncocytomas. Total RNA was extracted from 9 formalin-fixed paraffin embedded pituitary samples (4 primary, 3 recurrent oncocytomas and 2 normal tissues). Next-generation sequencing was performed for miRNA profiling. Transcriptome data of additional 6 samples' were obtained from NBCI GEO database for gene expression reanalysis and tissue-specific target prediction. Bioinformatical analysis, in vitro miRNA mimics transfection, luciferase reporter system and AlamarBlue assay were applied to characterize miRNA's function. 54 differentially expressed miRNAs and 485 genes in pituitary SCO vs. normal tissue and 8 miRNAs in recurrent vs. primary SCO were determined. Global miRNA downregulation and decreased level of DROSHA were detected in SCO samples vs. normal tissue. Transcriptome analysis revealed cell cycle alterations while miRNAs influenced mainly metabolic processes (tricarboxylic acid cycle-TCA, carbohydrate, lipid metabolism). Through miRNA-target interaction network the overexpressed Aconitase 2 potentially targeted by two downregulated miRNAs (miR-744-5p, miR-127-3p) was revealed. ACO2 and miR-744-5p interaction was validated by luciferase assay. MiR-127-3p and miR-744-5p significantly decreased cell proliferation in vitro. Our study firstly reported miRNA profile of pituitary oncocytoma. Our results suggest that tumor suppressor miRNAs may have an essential role in the pathogenesis of pituitary oncocytoma. Earlier reports showed downregulated TCA cycle in SCO which is extended by our results adding the role of miR-744-5p targeting ACO2.

MicroRNA Expression Profiling in Adrenal Myelolipoma.

Introduction: Adrenal myelolipoma (AML) is the second most common and invariably benign primary adrenal neoplasm. Due to the variable proportion of fat and hematopoietic elements and its often large size, it can cause differential diagnostic problems. Several reports confirmed the utility of miRNAs in the diagnosis of tumors, but miRNA expression in AML has not yet been investigated. Materials and Methods: Next-generation sequencing (NGS) was performed on 30 formalin-fixed, paraffin-embedded (FFPE) archived tissue samples [10 each of AML, adrenocortical adenoma (ACA), and adrenocortical carcinoma (ACC)]. Validation was performed by real-time quantitative reverse transcription polymerase chain reaction on a cohort containing 41 further FFPE samples (15 AML, 14 ACA, and 12 ACC samples). Circulating miRNA counterparts of significantly differentially expressed tissue miRNAs were studied in 33 plasma samples (11 each of ACA, ACC, and AML). Results: By NGS, 256 significantly differentially expressed miRNAs were discovered, and 8 of these were chosen for validation. Significant overexpression of hsa-miR-451a, hsa-miR-486-5p, hsa-miR-363-3p, and hsa-miR-150-5p was confirmed in AML relative to ACA and ACC. hsa-miR-184, hsa-miR-483-5p, and hsa-miR-183-5p were significantly overexpressed in ACC relative to ACA but not to AML. Circulating hsa-miR-451a and hsa-miR-363-3p were significantly overexpressed in AML, whereas circulating hsa-miR-483-5p and hsa-miR-483-3p were only significantly overexpressed in ACC vs ACA. Conclusions: We have found significantly differentially expressed miRNAs in AML and adrenocortical tumors. Circulating hsa-miR-451a might be a promising minimally invasive biomarker of AML. The lack of significantly different expression of hsa-miR-483-3p and hsa-miR-483-5p between AML and ACC might limit their applicability as diagnostic miRNA markers for ACC.

[The role of miRNAs in the pathogenesis of pituitary adenomas]. / A mikro-RNS-ek szerepe az agyalapimirigy-daganatok tumorbiológiájában.

MicroRNAs (miRNAs) are short, single stranded RNA molecules which play regulatory roles through posttranscriptional regulation of their target genes. Based on our current knowledge, more than 30% of the human protein-coding genes are regulated by miRNAs, hence influencing basic cellular mechanisms including cell proliferation, differentiation and cell death. Differential miRNA expression pattern has been detected in many different types of tumors and, recently, several publications have referred to miRNAs as potential therapeutic targets. Through adjustment of miRNA levels by artificial miRNAs administration or miRNA inhibition, we can influence not only one target gene but also complex biological pathways. Pituitary adenoma is the second most frequent intracranial tumor. In spite of this, the molecular mechanism of the pituitary adenoma formation is not yet entirely revealed. Recently, more and more evidences have been found suggesting that miRNAs have an important role in pituitary adenoma pathogenesis. Here, we summarize the recent results related to this role and highlight the therapeutic potentials in pituitary adenomas. Orv Hetil. 2018; 159(7): 252-259.

Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression.

BACKGROUND: Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored. METHODS: Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied. RESULTS: Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle. CONCLUSIONS: Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle.