MicroRNA in refined diagnosis of choroidal melanoma

Epigenetic studies of the level of microRNAs in human oncogenesis indicate their signifi cant role in the development and growth of malignant tumors of various origins. The fi rst works on the role of microRNAs in patients with uveal melanoma appeared in 2008.The aim: to analyze the expression level of miRNA-126 and miRNA-223 in the plasma blood of patients and to determine their signifi cance in the refi ned diagnosis of choroidal melanoma. Materials and methods. We examined 84 patients with choroidal melanoma (CM), mean age – 63.4 ± 1.2 (35–86 y.o.). Localization – a single CM node with a thickness of 0.77–17.19 mm. The control group consisted of 28 volunteers, age – 62.9 ± 1.42 (45–78 y.o.). Plasma miRNA expression levels were determined by real-time PCR.Results. An increase in the level of expression of miRNA-223 and miRNA-126 in blood plasma was confi rmed in all 84 patients with choroidal melanoma N0M0 compared with the control group. An increase in the expression of miRNA-223 and miRNA-126 was proved with an increase in tumor prominence.Conclusion. The obtained results of an increase in the expression of miRNA-223 indicate an increase in cell proliferation, and an increase in the expression of miRNA-126 on the activation of angiogenesis in a growing tumor, which makes it possible to recommend a study of the level of miRNA-223 and miRNA-126 for a more accurate diagnosis of small CM in cases of difficulty of differential diagnosis with other tumor-like diseases of the choroid.

Adiponectin Related Vascular and Cardiac Benefits in Obesity: Is There a Role for an Epigenetically Regulated Mechanism?

In obesity, several epigenetic modifications, including histones remodeling, DNA methylation, and microRNAs, could accumulate and determine increased expression of inflammatory molecules, the adipokines, that in turn might induce or accelerate the onset and development of cardiovascular and metabolic disorders. In order to better clarify the potential epigenetic mechanisms underlying the modulation of the inflammatory response by adipokines, the DNA methylation profile in peripheral leukocytes of the promoter region of IL-6 and NF-kB genes and plasma miRNA-21 levels were evaluated in 356 healthy subjects, using quantitative pyrosequencing-based analysis, and correlated with plasma adiponectin levels, body fat content and the primary pro-inflammatory markers. In addition, correlation analysis of DNA methylation profiles and miRNA-21 plasma levels with intima-media thickness (IMT), a surrogate marker for early atherosclerosis, left ventricular mass (LVM), left ventricular ejection fraction (LVEF), and cardiac performance index (MPI) was also performed to evaluate any potential clinical implication in terms of cardiovascular outcome. Results achieved confirmed the role of epigenetics in the obesity-related cardiovascular complications and firstly supported the potential role of plasma miRNA-21 and IL-6 and NF-kB DNA methylation changes in nucleated blood cells as potential biomarkers for predicting cardiovascular risk in obesity. Furthermore, our results, showing a role of adiponectin in preventing epigenetic modification induced by increased adipose tissue content in obese subjects, provide new evidence of an additional mechanism underlying the anti-inflammatory properties and the cardiovascular benefits of adiponectin. The exact mechanisms underlying the obesity-related epigenetic modifications found in the blood cells and whether similar epigenetic changes reflect adipose and myocardial tissue modifications need to be further investigated in future experiments.

BIOM-29. ASSOCIATION OF PLASMA microRNA-21 EXPRESSION WITH KARNOFSKY PERFORMANCE SCALE SCORES IN GLIOMA PATIENTS

Gliomas are one of the most common primary brain tumors. MicroRNA-21 (miRNA-21) has been shown in previous studies to be associated with prognosis in glioma patients. However, similar studies in the Asian population, particularly in Indonesia, are very limited. This study aimed to find the association of plasma miRNA-21 expression with functional status measured by Karnofsky Performance Scale (KPS) in Indonesian glioma patients. The patients were enrolled from a neuro-oncology referral center in Yogyakarta, Indonesia. MiRNA-21 expression from plasma was measured by real-time quantitative PCR. Clinical data were obtained from medical records. KPS scores were classified as low (< 70) and high (> 70). In total, 50 patients were included in this study. Most patients were diagnosed with WHO grade IV gliomas (30.4%), followed by grade II (30.4%), grade III (21.4%), and grade I (5.4%). Most patients (64%) have low KPS scores (< 70). Patients in the low KPS scores group have significantly higher miRNA-21 expression compared to patients in the high KPS scores group (2-∆CT 4.26 vs. 0.68, p=0.002). In conclusion, higher expression of plasma miRNA-21 is associated with worse functional status in glioma patients as measured by KPS.

Molecular diagnosis of polycystic ovary syndrome in obese and non-obese women by targeted plasma miRNA profiling

Objective Polycystic ovary syndrome (PCOS) is diagnosed based on the clinical signs, but its presentation is heterogeneous and potentially confounded by concurrent conditions, such as obesity and insulin resistance. miRNA have recently emerged as putative pathophysiological and diagnostic factors in PCOS. However, no reliable miRNA-based method for molecular diagnosis of PCOS has been reported. The aim of this study was to develop a tool for accurate diagnosis of PCOS by targeted miRNA profiling of plasma samples, defined on the basis of unbiased biomarker-finding analyses and biostatistical tools. Methods A case–control PCOS cohort was cross-sectionally studied, including 170 women classified into four groups: non-PCOS/lean, non-PCOS/obese, PCOS/lean, and PCOS/obese women. High-throughput miRNA analyses were performed in plasma, using NanoString technology and a 800 human miRNA panel, followed by targeted quantitative real-timePCR validation. Statistics were applied to define optimal normalization methods, identify deregulated biomarker miRNAs, and build classification algorithms, considering PCOS and obesity as major categories. Results The geometric mean of circulating hsa-miR-103a-3p, hsa-miR-125a-5p, and hsa-miR-1976, selected among 125 unchanged miRNAs, was defined as optimal reference for internal normalization (named mR3-method). Ten miRNAs were identified and validated after mR3-normalization as differentially expressed across the groups. Multinomial least absolute shrinkage and selection operator regression and decision-tree models were built to reliably discriminate PCOS vs non-PCOS, either in obese or non-obese women, using subsets of these miRNAs as performers. Conclusions We define herein a robust method for molecular classification of PCOS based on unbiased identification of miRNA biomarkers and decision-tree protocols. This method allows not only reliable diagnosis of non-obese women with PCOS but also discrimination between PCOS and obesity. Capsule We define a novel protocol, based on plasma miRNA profiling, for molecular diagnosis of PCOS. This tool not only allows proper discrimination of the condition in non-obese women but also permits distinction between PCOS and obesity, which often display overlapping clinical presentations.

Plasma-Derived microRNAs Are Influenced by Acute and Chronic Exercise in Patients With Heart Failure With Reduced Ejection Fraction

Background: Exercise training improves VO2peak in heart failure with reduced ejection fraction (HFrEF), but the effect is highly variable as it is dependent on peripheral adaptations. We evaluated changes in plasma-derived miRNAs by acute and chronic exercise to investigate whether these can mechanistically be involved in the variability of exercise-induced adaptations.Methods: Twenty-five male HFrEF patients (left ventricular ejection fraction < 40%, New York Heart Association class ≥ II) participated in a 15-week combined strength and aerobic training program. The effect of training on plasma miRNA levels was compared to 21 male age-matched sedentary HFrEF controls. Additionally, the effect of a single acute exercise bout on plasma miRNA levels was assessed. Levels of 5 miRNAs involved in pathways relevant for exercise adaptation (miR-23a, miR-140, miR-146a, miR-191, and miR-210) were quantified using RT-qPCR and correlated with cardiopulmonary exercise test (CPET), echocardiographic, vascular function, and muscle strength variables.Results: Expression levels of miR-146a decreased with training compared to controls. Acute exercise resulted in a decrease in miR-191 before, but not after training. Baseline miR-23a predicted change in VO2peak independent of age and left ventricular ejection fraction (LVEF). Baseline miR-140 was independently correlated with change in load at the respiratory compensation point and change in body mass index, and baseline miR-146a with change in left ventricular mass index.Conclusion: Plasma-derived miRNAs may reflect the underlying mechanisms of exercise-induced adaptation. In HFrEF patients, baseline miR-23a predicted VO2peak response to training. Several miRNAs were influenced by acute or repeated exercise. These findings warrant exploration in larger patient populations and further mechanistic in vitro studies on their molecular involvement.

Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency

Background Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. Methods The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. Results An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. Conclusion This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.

Associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation

The space radiation environment consists of multiple species of charged particles, including 28Si ions, that may impact brain function during and following missions. To develop biomarkers of the space radiation response, BALB/c and C3H female and male mice and their F2 hybrid progeny were irradiated with 28Si ions (350 MeV/n, 0.2 Gy) and tested for behavioral and cognitive performance 1, 6, and 12 months following irradiation. The plasma of the mice was collected for analysis of miRNA levels. Select pertinent brain regions were dissected for lipidomic analyses and analyses of levels of select biomarkers shown to be sensitive to effects of space radiation in previous studies. There were associations between lipids in select brain regions, plasma miRNA, and cognitive measures and behavioral following 28Si ion irradiation. Different but overlapping sets of miRNAs in plasma were found to be associated with cognitive measures and behavioral in sham and irradiated mice at the three time points. The radiation condition revealed pathways involved in neurodegenerative conditions and cancers. Levels of the dendritic marker MAP2 in the cortex were higher in irradiated than sham-irradiated mice at middle age, which might be part of a compensatory response. Relationships were also revealed with CD68 in miRNAs in an anatomical distinct fashion, suggesting that distinct miRNAs modulate neuroinflammation in different brain regions. The associations between lipids in selected brain regions, plasma miRNA, and behavioral and cognitive measures following 28Si ion irradiation could be used for the development of biomarker of the space radiation response.

Predict Colon Cancer by Pairing Plasma miRNAs: Establishment of a Normalizer-Free, Cross-Platform Model

BackgroundPlasma miRNAs are emerging biomarkers for colon cancer (CC) diagnosis. However, the lack of robust internal references largely limits their clinical application. Here we propose a ratio-based, normalizer-free algorithm to quantitate plasma miRNA for CC diagnosis.MethodsA miRNA-pair matrix was established by pairing differentially expressed miRNAs in the training group from GSE106817. LASSO regression was performed to select variables. To maximize the performance, four algorithms (LASSO regression, random forest, logistic regression, and SVM) were tested for each biomarker combination. Data from GSE106817 and GSE112264 were used for internal and external verification. RT-qPCR data acquired from another cohort were also used for external validation.ResultsAfter validation through four algorithms, we obtained a 4-miRNA pair model (miR-1246 miR-451a; miR-1246 miR-4514; miR-654-5p miR-575; miR-4299 miR-575) that showed good performance in differentiating CC from normal controls with a maximum AUC of 1.00 in internal verification and 0.93 in external verification. Tissue validation showed a maximum AUC of 0.81. Further external validation using RT-qPCR data exhibited good classifier ability with an AUC of 0.88.ConclusionWe established a cross-platform prediction model robust against sample-specific disturbance, which is not only well-performed in predicting CC but also promising in the diagnosis of other diseases.