Anting Liu Carlsen

Circulating microRNA expression profiles associated with systemic lupus erythematosus

OBJECTIVE To evaluate the specificity of expression patterns of cell-free circulating microRNAs (miRNAs) in systemic lupus erythematosus (SLE). METHODS Total RNA was purified from plasma, and 45 different specific, mature miRNAs were determined 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.

Changes in circulating microRNA-126 during treatment with chemotherapy and bevacizumab predicts treatment response in patients with metastatic colorectal cancer

Background:This study investigated the predictive value of circulating microRNA-126 (cir-miRNA-126) in patients with metastatic colorectal cancer (mCRC) treated with first-line chemotherapy combined with bevacizumab.Methods:The study included 68 patients. Blood samples (plasma) were collected before the treatment initiation, at the first clinical evaluation after 3 weeks and at progression. Levels of cir-miRNA-126 were determined by qRT–PCR after purification of total RNA from plasma. Primary clinical end points were response rates evaluated according to the Response Evaluation Criteria In Solid Tumours (RECIST) and progression-free survival (PFS).Results:Changes in circulating miRNA-126 during treatment were predictive of tumour response. Non-responding patients had a median increase in cir-miRNA-126 of 0.244 (95% confidence interval (CI), 0.050–0.565) compared with a median decrease of −0.374 (95% CI, −0.472 to −0.111) in the responding patients, P=0.002. A significant positive correlation was demonstrated by comparing the changes in tumour size with the changes in cir-miRNA-126, r=0.48, P=0.0001. Grouping the patients according to the changes in cir-miRNA-126 disclosed a borderline significant separation of the groups in the PFS analysis favouring patients with decreasing miRNA-126 levels, hazard ratio (HR) 0.60 (95% CI, 0.33–1.09), P=0.07.Conclusions:The present results indicate that changes in cir-miRNA-126 during treatment are related to the response to chemotherapy and bevacizumab in patients with mCRC, thus representing a possible biomarker for the resistance to anti-angiogenic containing treatments.

Free leptin index and PAPP‐A: a first trimester maternal serum screening test for pre‐eclampsia

Prophylaxis with low‐dose aspirin may reduce the risk of pre‐eclampsia (PE) if introduced in first trimester. The performance of first trimester maternal serum screening for PE using free leptin index (fLI) and PAPP‐A, where fLI = leptin/leptin soluble receptor was studied.

Specific autoantibody profiles and disease subgroups correlate with circulating micro-RNA in systemic sclerosis

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.

Circulating Extracellular microRNA in Systemic Autoimmunity

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.

Diurnal Variations of Human Circulating Cell-Free Micro-RNA

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.

Associations between gastrointestinal toxicity, micro RNA and cytokine production in patients undergoing myeloablative allogeneic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is a procedure with a high risk of treatment related mortality. The primary aim of the present study was to examine associations between markers of gastrointestinal toxicity, markers of systemic inflammation, and plasma levels of microRNA (miRNA) -155 and -146a during the first month after HSCT. The secondary aim was to characterize the impact of the toxic-inflammatory response on the function of circulating leukocytes during immune recovery. Thirty HSCT patients were included. Gastrointestinal injury was monitored by toxicity scores, lactulose-mannitol test and plasma citrulline, as a measure of the enterocyte population. Nadir of citrulline and maximum of oral toxicity scores, intestinal permeability, CRP and plasma levels of IL-6 and IL-10 was seen at day +7 post-HSCT. miRNA-155 and mi-RNA-146a showed an inverse relation with significantly elevated miRNA-155 and decreased miRNA-146a levels, from day 0 to day +28 compared with pre-conditioning levels. Citrulline levels below the median at day +7 were associated with higher spontaneous production of IL-6 and TNF-α as well as higher in vitro stimulated production of IL-17A at day +21. This study is the first to demonstrate that toxic responses to chemotherapy are accompanied by differential regulation of miRNAs with opposing effects on immune regulation. We find that a proinflammatory miRNA profile is sustained during the first three weeks after the transplantation, indicating that these miRNAs may play a role in the regulation of the inflammatory environment during immune reconstitution after HSCT.

MicroRNAs in cardiac arrhythmia: DNA sequence variation of MiR-1 and MiR-133A in long QT syndrome

Abstract 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.

Plasma MicroRNA Profiles in Patients with Early Rheumatoid Arthritis Responding to Adalimumab plus Methotrexate vs Methotrexate Alone: A Placebo-controlled Clinical Trial

Objective. The aim was to identify plasma (i.e., cell-free) microRNA (miRNA) predicting antitumor necrosis and/or methotrexate (MTX) treatment response in patients enrolled in an investigator-initiated, prospective, double-blinded, placebo-controlled trial (The OPERA study, NCT00660647). Methods. We included 180 disease-modifying antirheumatic drug–naive patients with early rheumatoid arthritis (RA) randomized to adalimumab (ADA; n = 89) or placebo (n = 91) in combination with MTX. Plasma samples before and 3 months after treatment initiation were analyzed for 91 specific miRNA by quantitative reverse transcriptase-polymerase chain reaction on microfluidic dynamic arrays. A linear mixed-effects model was used to test for associations between pretreatment miRNA and changes in miRNA expression and American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) Boolean (28 joints) remission at 3 and 12 months, applying false discovery rate correction for multiple testing. Using leave-one-out cross validation, we built predictive multivariate miRNA models and estimated classification performances using receiver-operating characteristics (ROC) curves. Results. In the ADA group, a higher pretreatment level of miR-27a-3p was significantly associated with remission at 12 months. The level decreased in remitting patients between pretreatment and 3 months, and increased in nonremitting patients. No associations were found in the placebo group receiving only MTX. Two multivariate miRNA models were able to predict response to ADA treatment after 3 and 12 months, with 63% and 82% area under the ROC curves, respectively. Conclusion. We identified miR-27a-3p as a potential predictive biomarker of ACR/EULAR remission in patients with early RA treated with ADA in combination with MTX. We conclude that pretreatment plasma-miRNA profiles may be of predictive value, but the results need confirmation in independent cohorts.

Placental protein-13 (PP13) in combination with PAPP-A and free leptin index (fLI) in first trimester maternal serum screening for severe and early preeclampsia

Abstract Background: Placental protein-13 (PP13) is involved in placental invasion and has been suggested as a maternal serum marker of preeclampsia (PE) development. However, the discriminatory ability of PP13 in first trimester has not been completely clarified. Methods: PP13 was measured in first trimester (week 10+3–13+6) maternal serum from 120 PE pregnancies and 267 control pregnancies and was correlated with clinical parameters. The population screening performance of PP13 in combination with the PE markers pregnancy associated plasma protein A (PAPPA) and free leptin index (fLI) was assessed by Monte Carlo simulation. Results: In severe PE (including HELLP) cases (n=26) the median PP13 concentration was 35.8 pg/mL (range: 17.8–85.5 pg/mL) and in PE pregnancies (n=10) with birth prior to week 34, the median PP13 concentration was 30.6 pg/mL (13.1–50.1 pg/mL), compared to controls with a median of 54.8 pg/mL (range: 15.4–142.6 pg/mL) (p<0.04). The population screening detection rate (DR) for a false-positive rate of 10% for severe PE and HELLP was 26% for PP13, 28% for PP13+PAPP-A, 33% for PP13+fLI, and 40% for PP13+PAPP-A+fLI. Conclusions: PP13 is a marker of severe PE and HELLP syndrome. The screening performance of PP13 can be markedly improved by combining it with fLI and PAPP-A.