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Objectives SSc-associated pulmonary arterial hypertension (SSc-APAH) is a late but devastating complication of SSc. Early identification of SSc-APAH may improve survival. We examined the role of circulating miRNAs in SSc-APAH. Methods Using quantitative RT-PCR the abundance of mature miRNAs in plasma was determined in 85 female patients with ACA-positive lcSSc. Twenty-two of the patients had SSc-APAH. Sixty-three SSc controls without PAH were matched for disease duration. Forty-six selected miRNA plasma levels were correlated with clinical data. Longitudinal samples were analysed from 14 SSc-APAH and 27 SSc patients. Results The disease duration was 12 years for the SSc-APAH patients and 12.7 years for the SSc controls. Plasma expression levels of 11 miRNAs were lower in patients with SSc-APAH. Four miRNAs displayed higher plasma levels in SSc-APAH patients compared with SSc controls. There was significant difference between groups for miR-20a-5p and miR-203a-3p when correcting for multiple comparisons (P = 0.002 for both). Receiver operating characteristics curve showed AUC = 0.69–0.83 for miR-21-5p and miR-20a-5p or their combination. miR-20a-5p and miR-203a-3p correlated inversely with NT-pro-Brain Natriuretic Protein levels (r = −0.42 and −0.47). Mixed effect model analysis could not identify any miRNAs as predictor of PAH development. However, miR-20a-5p plasma levels were lower in the longitudinal samples of SSc-APAH patients than in the SSc controls. Conclusions Our study links expression levels of the circulating plasma miRNAs, especially miR-20a-5p and miR-203a-3p, to the occurrence of SSc-APAH in female patients with ACA-positive lcSSc.

Objective: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a high risk of atherosclerosis and cardiovascular disease (CVD). MicroRNAs (miRNAs) are small non-coding RNAs that modulate protein translation, and dysregulation is seen in autoimmunity, atherosclerosis, and CVD. We investigate associations between circulating miRNAs and markers of atherosclerosis in SLE patients. Method: A group (n = 121) of well-characterized SLE patients were screened for atherosclerosis by cardiac computed tomography and carotid ultrasound. RNA was purified from plasma and 46 specific miRNAs were determined using quantitative real-time polymerase chain reaction. Results: Forty-one miRNAs were consistently detected. Fifty out of 118 available SLE patients had atherosclerosis. A profile consisting of three miRNAs (decreased miR-125b, miR-101, miR-375) was indicative of atherosclerosis. Multivariate logistic regression identified eight clinical manifestations associated with atherosclerotic outcome. The full classification profile showed a specificity of 88% and a sensitivity of 86%. Hierarchical clustering identified an eight-miRNA profile that differentiated a subgroup of SLE patients (n = 16) who had significantly increased venous thrombotic events (p = 0.045), a higher prevalence of β2-glycoprotein I antibodies (p = 0.029), and an increased prevalence of thrombocytopenia (p = 0.028). Conclusion: In this cross-sectional study, the circulating miRNA profile distinguished SLE patients with atherosclerosis from those without. Furthermore, an eight-miRNA signature was associated with thrombocytopenia, venous thrombotic events, and β2-glycoprotein I antibodies in SLE patients. Prospective studies are needed to confirm the findings and to establish the precise role of circulating miRNA profiling in the evaluation of atherosclerosis in SLE.

Aim: Neoadjuvant chemotherapy may represent a shift in the treatment of locally advanced colon cancer. The angiogenic couple has-microRNA-126 (miRNA-126) and epidermal growth factor-like domain 7 (EGFL7) are transcribed from the same gene and regulates all aspects of angiogenesis and may influence the ability of tumor cells to disseminate. The aim was to analyze the relationship between miRNA-126 and EGFL7 and disease recurrence in patients with locally advanced colon cancer treated with neoadjuvant chemotherapy. Methods: This study included 71 patients from a phase II study all planned for three cycles of capecitabine and oxaliplatin before surgery. Blood was sampled at baseline and right before and after the operation. Circulating miRNA-126 was analysed by RT-qPCR and a quantitative immunoassay was used for the analyses of EGFL7. Results: The rates of 5-year disease-free survival (DFS) and overall survival (OS) were 80% and 85%, respectively. The level of circulating miRNA-126 before the operation predicts recurrence, P = 0.035. In patients with values below and above the median the recurrence rate was 31% and 4%, respectively. Similar results applied to EGFL7. Awww.cdrjournal.com combined estimate identified a subgroup of patients (25 of 71) with no recurrence and a 5-year DFS and OS rate of 100%, respectively. Conclusion: MicroRNA-126 and EGFL7 are predictors for disease recurrence in patients with locally advanced colon cancer treated with neoadjuvant chemotherapy and may assist in selection of adjuvant chemotherapy.

BACKGROUND: MicroRNAs are small noncoding RNAs involved in the post-transcriptional regulation of protein synthesis. Extracellular microRNAs are accessible in a stable form in biofluids.OBJECTIVES: The aim was to identify individual microRNAs and/or subsets of microRNAs in CSF with biomarker potential and thus identify specific putative pathophysiological pathways.METHODS: In a two-step exploratory study design of PD, MSA, PSP, and controls, we initially profiled CSF microRNAs in a pilot cohort (n = 40) by screening for 372 microRNAs. Subsequently, we attempted to validate findings in an independent study cohort in CSF (n = 118) and ethylenediaminetetraacetic acid plasma (n = 114). This study cohort encompassed 46 microRNAs, of which 26 were singled out from the pilot cohort, and an additional 20 microRNAs were added based on previous publications. The most accurate diagnostic microRNA classifiers were identified in a multivariable logistic regression model adjusted for age and sex.RESULTS: A set of three microRNAs in CSF discriminated PD and MSA from controls with good diagnostic accuracy by receiver operating characteristics curve evaluation. The microRNAs were for PD versus controls: miR-7-5p, miR-331-5p, and miR-145-5p (area under the curve = 0.88) and MSA versus controls: miR-7-5p, miR-34c-3p, and miR-let-7b-5p (area under the curve = 0.87). The classifier that best distinguished MSA and PD consisted of two microRNAs: miR-9-3p and miR-106b-5p (area under the curve = 0.73). A single microRNA, miR-106b-5p, provided the best discrimination between PD and PSP (area under the curve = 0.85) in the CSF.CONCLUSIONS: Levels of specific trios of CSF-microRNAs discriminate well between α-synucleinopathies (PD and MSA) and controls. The results need to be validated in larger, independent cohorts. © 2018 International Parkinson and Movement Disorder Society.

Long QT syndrome (LQTS) is a genetic cardiac condition associated with prolonged ventricular repolarization, primarily a result of perturbations in cardiac ion channels, which predisposes individuals to life-threatening arrhythmias. Using DNA screening and sequencing methods, over 700 different LQTS-causing mutations have been identified in 13 genes worldwide. Despite this, the genetic cause of 30-50% of LQTS is presently unknown. MicroRNAs (miRNAs) are small (∼ 22 nucleotides) noncoding RNAs which post-transcriptionally regulate gene expression by binding complementary sequences within messenger RNAs (mRNAs). The human genome encodes over 1800 miRNAs, which target about 60% of human genes. Consequently, miRNAs are likely to regulate many complex processes in the body, indeed aberrant expression of various miRNA species has been implicated in numerous disease states, including cardiovascular diseases. MiR-1 and MiR-133A are the most abundant miRNAs in the heart and have both been reported to regulate cardiac ion channels. We hypothesized that, as a consequence of their role in regulating cardiac ion channels, genetic variation in the genes which encode MiR-1 and MiR-133A might explain some cases of LQTS. Four miRNA genes (miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2), which encode MiR-1 and MiR-133A, were sequenced in 125 LQTS probands. No genetic variants were identified in miR-1-1 or miR-133a-1; but in miR-1-2 we identified a single substitution (n.100A> G) and in miR-133a-2 we identified two substitutions (n.-19G> A and n.98C> T). None of the variants affect the mature miRNA products. Our findings indicate that sequence variants of miR-1-1, miR-1-2, miR-133a-1 and miR-133a-2 are not a cause of LQTS in this cohort.

BackgroundOvarian cancer is the fifth most common cancer in women worldwide. Moreover, there are no reliable minimal invasive tests to secure the diagnosis of malignant pelvic masses. Cell-free, circulating microRNAs have the potential as diagnostic biomarkers in cancer. Here, we performed and validated a miRNA panel with the potential to distinguish OC from benign pelvic masses.MethodsThe profile of plasma microRNA was determined with a panel of 46 candidates in a discovery group and a validation group, each consisting of 190 pre-surgery plasma samples from age-matched patients with malignant (n = 95) and benign pelvic mass (n = 95), by real time RT-qPCR.ResultsFour up-regulated (miR-200c-3p, miR-221-3p, miR-21-5p, and miR-484) and two down-regulated (miR-195-5p and miR-451a) microRNAs were discovered. From those, miR-200c-3p and miR-221-3p were further confirmed in a validation cohort. A combination of these 2 microRNAs together with CA-125 yielded an overall diagnostic accuracy of AUC = 0.96.ConclusionsWe showed consistent plasma microRNA profiles that provide independent diagnostic information of late stage OC.

There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles and PDAC in a disease and disease-control cohort.We performed a microarray profiling of 847 different mature miRNAs from plasma in an exploratory cohort of 20 patients with PDAC or other pancreatic diseases, profiling of 45 miRNAs in plasma samples from PDAC (n = 48) and disease controls (n = 47), and evaluation of associations of data with diagnosis, survival, and CA-19-9.We find 7 significantly deregulated miRNAs in PDAC using univariate statistics. At a false-discovery rate of 5%, miRNA-375 remained significantly elevated in PDAC. MicroRNA-375 did not improve diagnosis of PDAC in this cohort (70% accuracy) and did not correlate with survival. However, 3 controls (other gastrointestinal cancers) with increased CA-19-9 did not show increased miRNA-375.In the plasma-miRNA population, we find miRNA-375, which is selectively expressed in the endocrine pancreas under normal conditions, increased in PDAC cases compared with patients with other pancreatic or gastrointestinal diseases. The miRNA-375 does not outperform CA-19-9 diagnostically in the present cohort. However, it shows promising specificity and should be examined in larger prospective studies.

A 24-hour light and dark cycle-dependent rhythmicity pervades physiological processes in virtually all living organisms including humans. These regular oscillations are caused by external cues to endogenous, independent biological time-keeping systems (clocks). The rhythm is reflected by gene expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples. An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA) that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation of circadian gene expression. Here, we investigate 92 miRNAs in plasma samples from 24 young male, healthy volunteers repeatedly sampled 9 times during a 24-hour stay in a regulated environment. We demonstrate that a third (26/79) of the measurable plasma miRNAs (using RT-qPCR on a microfluidic system) exhibit a rhythmic behavior and are distributed in two main phase patterns. Some of these miRNAs weakly target known clock genes and many have strong targets in intracellular MAPK signaling pathways. These novel findings highlight the importance of considering bio-oscillations in miRNA biomarker studies and suggest the further study of a set of specific circulating miRNAs in the regulation and functioning of biological clocks.

Objective.To evaluate the expression profile of cell-free circulating microRNA (miRNA) in systemic sclerosis (SSc), healthy controls (HC), and systemic lupus erythematosus (SLE).Methods.Total RNA was purified from plasma and 45 different, mature miRNA were measured using quantitative PCR assays after reverse transcription. Samples (n = 189) were from patients with SSc (n = 120), SLE (n = 29), and from HC (n = 40). Expression data were clustered by principal components analysis, and diagnostically specific miRNA profiles were developed by leave-one-out cross-validation. Diagnostic probability scores were derived from stepwise logistic regression.Results.Thirty-seven miRNA specificities were consistently detected and 26 of these were unaffected by SSc sample age and present in more than two-thirds of SSc samples. SSc cases showed a distinct expression profile with 14/26 miRNA significantly decreased (false discovery rate < 0.05) and 5/26 increased compared with HC. A 21-miRNA classifier gave optimum accuracy (80%) for discriminating SSc from both HC and SLE. The discrimination between HC and SSc (95% accuracy) was strongly driven by miRNA of the 17∼92 cluster and by miR-16, -223, and -638, while SLE and SSc differed mainly in the expression of miR-142-3p, -150, -223, and -638. Except for a weak correlation between anti-Scl-70 and miR-638 (p = 0.048), there were no correlations with other patient variables.Conclusion.Circulating miRNA profiles are characteristic for SSc compared with both HC and SLE cases. Some of the predicted targets of the differentially regulated miRNA are of relevance for transforming growth factor-β signaling and fibrosis, but need to be validated in independent studies.

MicroRNAs (miRNAs) are differentially regulated in healthy, activated, inflamed, neoplastic, or otherwise pathological cells and tissues. While their main functions are executed intracellularly, many miRNAs can reproducibly be detected extracellularly in plasma and serum. This circulating, extracellular miRNA is protected against degradation by complexation with carrier proteins and/or by being enclosed in subcellular membrane vesicles. This, together with their tissue- and disease-specific expression, has fuelled the interest in using circulating microRNA profiles as harbingers of disease, i.e., as diagnostic analytes and as clues to dysregulated pathways in disease. Many studies show that inflammation and immune dysregulation, e.g., in autoimmune diseases, are associated with distinct miRNA expression changes in targeted tissues and in innate and adaptive immunity cells such as lymphocytes, natural killer cells, neutrophil granulocytes, and monocyte-macrophages. Exploratory studies (only validated in a few cases) also show that specific profiles of circulating miRNAs are associated with different systemic autoimmune diseases including systemic lupus erythematosus (SLE), systemic sclerosis, and rheumatoid arthritis. Even though the link between cellular alterations and extracellular profiles is still unpredictable, the data suggest that circulating miRNAs in autoimmunity may become diagnostically useful. Here, we review important circulating miRNAs in animal models of inflammation and in systemic autoimmunity and summarize some proposed functions of miRNAs in immune regulation and dysregulation. We conclude that the studies suggest new hypotheses and additional experiments, and that further diagnostic development is highly dependent on analytical method development and on obtaining sufficient numbers of uniformly processed samples from clinically well-characterized patients and controls.

Objective. To evaluate the expression profiles of cell-free plasma miRNAs in SSc and to characterize their correlation with disease subgroups (lcSSc and dcSSc) and with autoantibody profiles.Methods. Using quantitative RT-PCR, the abundance of 45 mature miRNAs in plasma was determined in 95 patients (lcSSc = 63; dcSSc = 32), representing the following autoantibody subgroups: ACA, anti-DNA topoisomerase I, anti-RNA polymerase III and anti-U1-ribonucleoprotein. MiRNA data were correlated with clinical and paraclinical data. Multiple regression was used to model membership of the lcSSc, dcSSc and autoantibody subgroups, based on miRNA expression profiles.Results. Thirty-six miRNAs were measurable in all samples. Four (miRNA-223, -181b, -342-3p and -184) were differently expressed in lcSSc and dcSSc (false discovery rate < 0.05). Ten miRNAs exhibited statistically significantly different levels in one or more autoantibody groups, and five (miRNA-409, -184, -92a, -29a and -101) remained significant after correction for multiple comparisons. Multiple regression models accurately predicted ACA and anti-DNA topoisomerase I antibody-positive patients (area under the curve (AUC) = 0.97 and 0.93, respectively) as well as membership of the dcSSc and lcSSc groups (AUC = 0.88).Conclusion. Circulating miRNA profiles differ between lcSSc and dcSSc patients and between patients with different autoantibodies. This is the first time autoantibody profiles, disease phenotypes and plasma miRNA profiles have been shown to correlate in an autoimmune disease. The data support a pathobiological role of miRNAs because specific miRNAs associate with autoantibody profiles of known diagnostic and prognostic value.

OBJECTIVE. To evaluate the specificity of expression patterns of cell-free circulating microRNAs (mi-RNAs) in systemic lupus erythematosus (SLE). METHODS. Total RNA was purified from plasma, and 45 different specific, mature miRNAs were deter-mined using quantitative reverse transcription–polymerase chain reaction assays. A total of 409 plasma samples were obtained from 364 different patients with SLE, healthy control subjects, and control subjects with other autoimmune diseases. The results in the primary cohort of 62 patients with SLE and 29 healthy control subjects were validated in 2 independent cohorts: a validation cohort comprising 68 patients with SLE and 68 healthy control subjects, and a disease control cohort comprising 20 patients with SLE (19 of whom were from the other validation cohort), 46 healthy control subjects, 38 patients with vasculitis, 18 patients with rheumatoid arthritis, and 20 immunosuppressed patients. RESULTS. Seven miRNAs were statistically significantly differentially expressed in plasma from patients with SLE. The expression of miRNA-142–3p (miR-142–3p) and miR-181a was increased, and the expression of miR-106a, miR-17, miR-20a, miR-203, and miR-92a was decreased. In addition, the expression of miR-342–3p, miR-223, and miR-20a was significantly decreased in SLE patients with active nephritis. A predictive model for SLE based on 2 or 4 miRNAs differentiated patients with SLE from control subjects (76% accuracy) when validated independently (P < 2 × 10(−9)). Use of the 4-miRNA model provided highly significant differentiation between the SLE group and disease controls, except for those with vasculitis. CONCLUSION. Circulating miRNAs are systematically altered in SLE. A 4-miRNA signature was diagnostic of SLE, and a specific subset of miRNA profiles was associated with nephritis. All of the signature miRNAs target genes in the transforming growth factor β signaling pathways. Other targets include regulation of apoptosis, cytokine–cytokine receptors, T cell development, and cytoskeletal organization. These findings highlight possible dysregulated pathways in SLE and suggest that circulating miRNA patterns distinguish SLE from other immunoinflammatory phenotypes.