First Trimester Screening of Circulating C19MC microRNAs Can Predict Subsequent Onset of Gestational Hypertension

Objective

The objective of the study was to evaluate risk assessment for gestational hypertension based on the profile of circulating placental specific C19MC microRNAs in early pregnancy.

Study design

The prospective longitudinal cohort study of women enrolled at first trimester screening at 10 to 13 weeks was carried out (n = 267). Relative quantification of placental specific C19MC microRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) was determined in 28 normal pregnancies and 18 pregnancies which developed gestational hypertension using real-time PCR and a comparative Ct method relative to synthetic C. elegans microRNA (cel-miR-39).

Results

Increased extracellular C19MC microRNA plasmatic levels (miR-516-5p, p<0.001; miR-517*, p = 0.007; miR-520h, p<0.001; miR-518b, p = 0.002) were detected in patients destined to develop gestational hypertension. MiR-520h had the best predictive performance with a PPV of 84.6% at a 7.1% false positive rate. The combination of miR-520h and miR-518b was able to predict 82.6% of women at the same false positive rate. The overall predictive capacity of single miR-518b (73.3% at 14.3% FPR), miR-516-5p (70.6% at 17.9% FPR) and miR-517* (57.9% at 28.6% FPR) biomarkers was lower.

Conclusion

The study brought interesting finding that the up-regulation of miR-516-5p, miR-517*, miR-520h and miR-518b is associated with a risk of later development of gestational hypertension. First trimester screening of extracellular miR-520h alone or in combination with miR-518b identified a significant proportion of women with subsequent gestational hypertension.     

Curated MicroRNAs in Urine and Blood Fail to Validate as Predictive Biomarkers for High-Risk Prostate Cancer

Purpose

The purpose of this study was to determine if microRNA profiling of urine and plasma at radical prostatectomy can distinguish potentially lethal from indolent prostate cancer.

Materials and Methods

A panel of microRNAs was profiled in the plasma of 70 patients and the urine of 33 patients collected prior to radical prostatectomy. Expression of microRNAs was correlated to the clinical endpoints at a follow-up time of 3.9 years to identify microRNAs that may predict clinical response after radical prostatectomy. A machine learning approach was applied to test the predictive ability of all microRNAs profiled in urine, plasma, and a combination of both, and global performance assessed using the area under the receiver operator characteristic curve (AUC). Validation of urinary expression of miRNAs was performed on a further independent cohort of 36 patients.

Results

The best predictor in plasma using eight miRs yielded only moderate predictive performance (AUC = 0.62). The best predictor of high-risk disease was achieved using miR-16, miR-21 and miR-222 measured in urine (AUC = 0.75). This combination of three microRNAs in urine was a better predictor of high-risk disease than any individual microRNA. Using a different methodology we found that this set of miRNAs was unable to predict high-volume, high-grade disease.

Conclusions

Our initial findings suggested that plasma and urinary profiling of microRNAs at radical prostatectomy may allow prognostication of prostate cancer behaviour. However we found that the microRNA expression signature failed to validate in an independent cohort of patients using a different platform for PCR. This highlights the need for independent validation patient cohorts and suggests that urinary microRNA signatures at radical prostatectomy may not be a robust way to predict the course of clinical disease after definitive treatment for prostate cancer.     

Decreased Serum Level of miR-146a as Sign of Chronic Inflammation in Type 2 Diabetic Patients

Background

There is increasing evidence that chronic inflammation is an important determinant in insulin resistance and in the pathogenesis of type 2 diabetes (T2D). MicroRNAs constitute a newly discovered system of cell regulation and in particular two microRNAs (miR-146a and miR-155) have been described as regulators and biomarkers of inflammation.

Aim

To determine a putative association between the levels of miR-146a and miR-155 in serum of T2D patients, clinical parameters and serological indicators of inflammation.

Methods

We performed quantitative Real Time PCR (qPCR) of microRNAs from serum (56 Ecuadorian T2D ambulatory patients and 40 non-diabetic controls). In addition, we evaluated T2D-related serum cytokines.chemokines and growth factors using a commercially available multi-analyte cytometric bead array system. We correlated outcomes to clinical parameters, including BMI, HbA1c and lipid state.

Results

The Ecuadorian non-diabetic controls appeared as overweight (BMI>25: patients 85%, controls 82.5%) and as dyslipidemic (hypercholesterolemia: patients 60.7%, controls 67.5%) as the patients.

• The serum levels of miR-146a were significantly reduced in T2D patients as compared to these non-diabetic, but obese/dyslipidemic control group (mean patients 0.61, mean controls set at 1; p = 0.042), those of miR-155 were normal.
• The serum levels of both microRNAs correlated to each other (r = 0.478; p<0.001) and to leptin levels. The microRNAs did not correlate to BMI, glycemia and dyslipidemia.
• From the tested cytokines, chemokines and growth factors, we found IL-8 and HGF significantly raised in T2D patients versus non-diabetic controls (p = 0.011 and 0.023 respectively).

Conclusions

This study shows decreased serum anti-inflammatory miR-146a, increased pro-inflammatory IL-8 and increased HGF (a vascular/insular repair factor) as discriminating markers of failure of glucose control occurring on the background of obesity and dyslipidemia.     

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose

We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).

Experimental Design

Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.

Results

In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.

Conclusions

We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.     

Urinary miR-29 Correlates with Albuminuria and Carotid Intima-Media Thickness in Type 2 Diabetes Patients

Background

Cell-free microRNAs stably and abundantly exist in body fluids and emerging evidence suggests cell-free microRNAs as novel and non-invasive disease biomarker. Deregulation of miR-29 is involved in the pathogenesis of diabetic nephropathy and insulin resistance thus may be implicated in diabetic vascular complication. Therefore, we investigated the possibility of urinary miR-29 as biomarker for diabetic nephropathy and atherosclerosis in patients with type 2 diabetes.

Methods

83 patients with type 2 diabetes were enrolled in this study, miR-29a, miR-29b and miR-29c levels in urine supernatant was determined by TaqMan qRT-PCR, and a synthetic cel-miR-39 was added to the urine as a spike-in control before miRNAs extraction. Urinary albumin excretion rate and urine albumin/creatinine ratio, funduscopy and carotid ultrasound were used for evaluation of diabetic vascular complication. The laboratory parameters indicating blood glucose level, renal function and serum lipids were also collected.

Results

Patients with albuminuria (n = 42, age 60.62±12.00yrs) showed significantly higher comorbidity of diabetic retinopathy (p = 0.015) and higher levels of urinary miR-29a (p = 0.035) compared with those with normoalbuminuria (n = 41, age 58.54±14.40yrs). There was no significant difference in urinary miR-29b (p = 0.148) or miR-29c level (p = 0.321) between groups. Urinary albumin excretion rate significantly correlated with urinary miR-29a level (r = 0.286, p = 0.016), while urinary miR-29b significantly correlated with carotid intima-media thickness (cIMT) (r = 0.286, p = 0.046).

Conclusion

Urinary miR-29a correlated with albuminuria while urinary miR-29b correlated with carotid intima-media thickness (cIMT) in patients with type 2 diabetes. Therefore, they may have the potential to serve as alternative biomarker for diabetic nephropathy and atherosclerosis in type 2 diabetes.     

Integrated miRNA-mRNA Analysis Revealing the Potential Roles of miRNAs in Chordomas

Introduction

Emerging evidence suggests that microRNAs (miRNAs) are crucially involved in tumorigenesis and that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision. However, no studies have applied integrated analysis to miRNA and mRNA profiles in chordomas. The purpose of this study was to provide insights into the pathogenesis of chordomas by using this integrated analysis method.

Methods

Differentially expressed miRNAs and mRNAs of chordomas (n = 3) and notochord tissues (n = 3) were analyzed by using microarrays with hierarchical clustering analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Then, GO and pathway analyses were performed for the intersecting genes.

Results

The microarray analysis indicated that 33 miRNAs and 2,791 mRNAs were significantly dysregulated between the two groups. Among the 2,791 mRNAs, 911 overlapped with putative miRNA target genes. A pathway analysis showed that the MAPK pathway was consistently enriched in the chordoma tissue and that miR-149-3p, miR-663a, miR-1908, miR-2861 and miR-3185 likely play important roles in the regulation of MAPK pathways. Furthermore, the Notch signaling pathway and the loss of the calcification or ossification capacity of the notochord may also be involved in chordoma pathogenesis.

Conclusion

This study provides an integrated dataset of the miRNA and mRNA profiles in chordomas, and the results demonstrate that not only the MAPK pathway and its related miRNAs but also the Notch pathway may be involved in chordoma development. The occurrence of chordoma may be associated with dysfunctional calcification or ossification of the notochord.     

Plasma MicroRNAs as Potential Noninvasive Biomarkers for In-Stent Restenosis

Objective

To investigate whether microRNAs (miRs) can serve as novel biomarkers for in-stent restenosis (ISR).

Methods

This retrospective, observational single-centre study was conducted at the cardiovascular department of a tertiary hospital centre in the north of China. Follow-up coronary angiography at 6 to 12 months was performed in 181 consecutive patients implanted with drug-eluting stents. Fifty-two healthy volunteers served as the control group. The plasma miRs levels were analyzed by quantitative real-time PCR. Receiver-operating characteristic curve (ROC) analysis was performed to investigate the characters of these miRs as potential biomarkers of ISR.

Results

MiR-21 levels in ISR patients were significantly higher than those in non-ISR patients and healthy controls (P<0.05), while miR-100 (P<0.05), miR-143 (P<0.001) and miR-145 (P<0.0001) levels were significantly decreased in ISR patients. Further analysis showed that miR-21 levels were remarkably increased (P = 0.045), while miR-100 (P = 0.041), miR-143 (P = 0.029) and miR-145 (P<0.01) levels were dramatically decreased in patients with diffuse ISR compared to those with focal ISR. ROC analysis demonstrated that the area under curve of miR-145, miR-143, miR-100 and miR-21 were 0.880 (95% confidence interval; CI = 0.791–0.987, P<0.001), 0.818 (95% confidence interval; CI = 0.755–0.963, P<0.001), 0.608 (95% confidence interval; CI = 0.372–0.757, P<0.05) and 0.568 (95% confidence interval; CI = 0.372–0.757, P<0.05), with specificity of 83.1%, 80.1%, 68.9% and 68.6%, and sensitivity of 88.7%, 82.1%, 60.2% and 50.1%, respectively.

Conclusions

Circulating miR-143 and miR-145 levels are associated with the occurrence of ISR and can serve as novel noninvasive biomarkers for ISR.     

MicroRNAs in renal cell carcinoma: diagnostic implications of serum miR-1233 levels

Background

MicroRNA expression is altered in cancer cells, and microRNAs could serve as diagnostic/prognostic biomarker for cancer patients. Our study was designed to analyze circulating serum microRNAs in patients with renal cell carcinoma (RCC).

Methodology/Principal Findings

We first explored microRNA expression profiles in tissue and serum using TaqMan Low Density Arrays in each six malignant and benign samples: Although 109 microRNAs were circulating at higher levels in cancer patients'' serum, we identified only 36 microRNAs with up-regulation in RCC tissue and serum of RCC patients. Seven candidate microRNAs were selected for verification based on the finding of up-regulation in serum and tissue of RCC patients: miR-7-1*, miR-93, miR-106b*, miR-210, miR-320b, miR-1233 and miR-1290 levels in serum of healthy controls (n = 30) and RCC (n = 33) patients were determined using quantitative real-time PCR (TaqMan MicroRNA Assays). miR-1233 was increased in RCC patients, and thus validated in a multicentre cohort of 84 RCC patients and 93 healthy controls using quantitative real-time PCR (sensitivity 77.4%, specificity 37.6%, AUC 0.588). We also studied 13 samples of patients with angiomyolipoma or oncocytoma, whose serum miR-1233 levels were similar to RCC patients. Circulating microRNAs were not correlated with clinical-pathological parameters.

Conclusions/Significance

MicroRNA levels are distinctly increased in cancer patients, although only a small subset of circulating microRNAs has a tumor-specific origin. We identify circulating miR-1233 as a potential biomarker for RCC patients. Larger-scaled studies are warranted to fully explore the role of circulating microRNAs in RCC.     

Stromal Expression of MiR-21 Predicts Biochemical Failure in Prostate Cancer Patients with Gleason Score 6

Aim

microRNAs (miRNAs) are involved in various neoplastic diseases, including prostate cancer (PCs). The aim of this study was to investigate the miRNA profile in PC tissue, to assess their association with clinicopathologic data, and to evaluate the potential of miRNAs as diagnostic and prognostic markers.

Materials and Methods

From a cohort of 535 patients submitted to radical prostatectomy (RP), a sample of 30 patients (14 patients with rapid biochemical failure (BF) and 16 patients without BF) with Gleason score 7 were analyzed. A total of 1435 miRNAs were quantified by microarray hybridization, and selected miRNAs with the highest Standard deviation (n = 50) were validated by real-time quantitative PCR (qRT-PCR). In situ hybridization (ISH) was used to evaluate the expression of miR-21.

Results

miR-21 was the only miR that was significantly up-regulated in the BF group (p = 0.045) miR-21 was up-regulated in patients with BF compared with non-BF group (p = 0.05). In univariate analyses, high stromal expression of miR-21 had predictive impact on biochemical failure-free survival (BFFS) and clinical failure-free survival (CFFS) (p = 0.006 and p = 0.04, respectively). In the multivariate analysis, high stromal expression of miR-21 expression was found to be an independent prognostic factor for BFFS in patients with Gleason score 6 (HR 2.41, CI 95% 1.06–5.49, p = 0.037).

Conclusion

High stromal expression of miR-21 was associated with poor biochemical recurrence-free survival after RP. For patients with Gleason score 6, miR-21 may help predict the risk of future disease progression and thereby help select patients for potential adjuvant treatment or a more stringent follow-up.     

Prognostic Role of MicroRNA-181a/b in Hematological Malignancies: A Meta-Analysis

Background

Emerging evidence has shown that miRNAs participate in human carcinogenesis as tumor suppressors or oncogenes, and have prognostic value for patients with cancers. In recent years, the miR-181 family was found dysregulated in a variety of human cancers and significantly associated with clinical outcome of cancerous patients. MiR-181a and miR-181b (miR-181a/b) were the most investigated members in the family. However, the results of miR-181a/b from different studies were inconsistent. Therefore, we performed a meta-analysis to summarize all the results from available studies, aiming to delineate the prognostic role of miR-181a/b in human cancers.

Methods

The identified articles were retrieved from the two main on-line databases, PubMed and EMBASE. We extracted and estimated the hazard ratios (HRs) for overall survival (OS), which compared the high and low expression levels of miR-181a/b in patients of the available studies. Each individual HR was used to calculate the pooled HR.

Results

Eleven studies of 1252 patients were selected into the final meta-analysis after a strict filtering and qualifying process. Fixed model or random model method was chosen depending on the heterogeneity between the studies. The subgroup analysis showed that high expressed miR-181a/b could prolong OS in patients with hematological malignancies rather than low expression level (HR = 0.717, P<0.0001). But the expression of miR-181a/b was not significantly relative to OS in patients with various cancers (HR = 0.861, p = 0.356).

Conclusion

Our study indicates that the expression level of miR-181a/b is significantly associated with OS in hematological malignancies and can be an important clinical prognostic factor for those patients.     

Prognostic significance of miR-205 in endometrial cancer

Purpose

microRNAs have emerged as key regulators of gene expression, and their altered expression has been associated with tumorigenesis and tumor progression. Thus, microRNAs have potential as both cancer biomarkers and/or potential novel therapeutic targets. Although accumulating evidence suggests the role of aberrant microRNA expression in endometrial carcinogenesis, there are still limited data available about the prognostic significance of microRNAs in endometrial cancer. The goal of this study is to investigate the prognostic value of selected key microRNAs in endometrial cancer by the analysis of archival formalin-fixed paraffin-embedded tissues.

Experimental Design

Total RNAs were extracted from 48 paired normal and endometrial tumor specimens using Trizol based approach. The expression of miR-26a, let-7g, miR-21, miR-181b, miR-200c, miR-192, miR-215, miR-200c, and miR-205 were quantified by real time qRT-PCR expression analysis. Targets of the differentially expressed miRNAs were quantified using immunohistochemistry. Statistical analysis was performed by GraphPad Prism 5.0.

Results

The expression levels of miR-200c (P<0.0001) and miR-205 (P<0.0001) were significantly increased in endometrial tumors compared to normal tissues. Kaplan-Meier survival analysis revealed that high levels of miR-205 expression were associated with poor patient overall survival (hazard ratio, 0.377; Logrank test, P = 0.028). Furthermore, decreased expression of a miR-205 target PTEN was detected in endometrial cancer tissues compared to normal tissues.

Conclusion

miR-205 holds a unique potential as a prognostic biomarker in endometrial cancer.     

Concordant Changes of Plasma and Kidney MicroRNA in the Early Stages of Acute Kidney Injury: Time Course in a Mouse Model of Bilateral Renal Ischemia-Reperfusion

Background

Acute kidney injury (AKI) is a syndrome characterized by the rapid loss of the kidney excretory function and is strongly associated with increased early and long-term patient morbidity and mortality. Early diagnosis of AKI is challenging; therefore we profiled plasma microRNA in an effort to identify potential diagnostic circulating markers of renal failure. The goal of the present study was to investigate the dynamic relationship of circulating and renal microRNA profiles within the first 24 hours after bilateral ischemia-reperfusion kidney injury in mice.

Methodology/Principal Findings

Bilateral renal ischemia was induced in C57Bl/6 mice (n = 10 per group) by clamping the renal pedicle for 27 min. Ischemia-reperfusion caused highly reproducible, progressive, concordant elevation of miR-714, miR-1188, miR-1897-3p, miR-877*, and miR-1224 in plasma and kidneys at 3, 6 and 24 hours after acute kidney injury compared to the sham-operated mice (n = 5). These dynamics correlated with histologic findings of kidney injury and with a conventional plasma marker of renal dysfunction (creatinine). Pathway analysis revealed close association between miR-1897-3p and Nucks1 gene expression, which putative downstream targets include genes linked to renal injury, inflammation and apoptosis.

Conclusions/Significance

Systematic profiling of renal and plasma microRNAs in the early stages of experimental AKI provides the first step in advancing circulating microRNAs to the level of promising novel biomarkers.     

miR-141 and miR-200c as Markers of Overall Survival in Early Stage Non-Small Cell Lung Cancer Adenocarcinoma

Background

Several treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established.

Methods

miRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44.

Results

High miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma – but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001).

Conclusion

High miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis.     

Conjunctival MicroRNA Expression in Inflammatory Trachomatous Scarring

Purpose

Trachoma is a fibrotic disease of the conjunctiva initiated by Chlamydia trachomatis infection. This blinding disease affects over 40 million people worldwide yet the mechanisms underlying its pathogenesis remain poorly understood. We have investigated host microRNA (miR) expression in health (N) and disease (conjunctival scarring with (TSI) and without (TS) inflammation) to determine if these epigenetic differences are associated with pathology.

Methods

We collected two independent samples of human conjunctival swab specimens from individuals living in The Gambia (n = 63 & 194). miR was extracted, and we investigated the expression of 754 miR in the first sample of 63 specimens (23 N, 17 TS, 23 TSI) using Taqman qPCR array human miRNA genecards. Network and pathway analysis was performed on this dataset. Seven miR that were significantly differentially expressed between different phenotypic groups were then selected for validation by qPCR in the second sample of 194 specimens (93 N, 74 TS, 22 TSI).

Results

Array screening revealed differential expression of 82 miR between N, TS and TSI phenotypes (fold change >3, p<0.05). Predicted mRNA targets of these miR were enriched in pathways involved in fibrosis and epithelial cell differentiation. Two miR were confirmed as being differentially expressed upon validation by qPCR. miR-147b is significantly up-regulated in TSI versus N (fold change = 2.3, p = 0.03) and miR-1285 is up-regulated in TSI versus TS (fold change = 4.6, p = 0.005), which was consistent with the results of the qPCR array.

Conclusions

miR-147b and miR-1285 are up-regulated in inflammatory trachomatous scarring. Further investigation of the function of these miR will aid our understanding of the pathogenesis of trachoma.     

Genome-Wide Sequencing of Cellular microRNAs Identifies a Combinatorial Expression Signature Diagnostic of Sepsis

Rationale

Sepsis is a common cause of death in the intensive care unit with mortality up to 70% when accompanied by multiple organ dysfunction. Rapid diagnosis and the institution of appropriate antibiotic therapy and pressor support are therefore critical for survival. MicroRNAs are small non-coding RNAs that play an important role in the regulation of numerous cellular processes, including inflammation and immunity.

Objectives

We hypothesized changes in expression of microRNAs during sepsis may be of diagnostic value in the intensive care unit (ICU).

Methods

Massively parallel sequencing of microRNAs was utilised for screening microRNA candidates. Putative microRNAs were validated using quantitative real-time PCR (qRT-PCR). This study includes data from both a training cohort (UK) and an independent validation cohort (Sweden). A linear discriminant statistical model was employed to construct a diagnostic microRNA signature.

Results

A panel of known and novel microRNAs were detectable in the blood of patients with sepsis. After qRT-PCR validation, microRNA miR-150 and miR-4772-5p-iso were able to discriminate between patients who have systemic inflammatory response syndrome and patients with sepsis. This finding was also validated in independent cohort with an average diagnostic accuracy of 86%. Fractionating the cellular components of blood reveals miR-4772-5p-iso is expressed differentially in monocytes. Functional experiments using primary human monocytes demonstrate that it expressed in response to TLR ligation.

Conclusions

Taken together, these data provide a novel microRNA signature of sepsis that should allow rapid point-of-care diagnostic assessment of patients on ICU and also provide greater insight into the pathobiology of this severe disease.     

A Panel of 4 microRNAs Facilitates the Prediction of Left Ventricular Contractility after Acute Myocardial Infarction

Background

Prediction of clinical outcome after acute myocardial infarction (AMI) is challenging and would benefit from new biomarkers. We investigated the prognostic value of 4 circulating microRNAs (miRNAs) after AMI.

Methods

We enrolled 150 patients after AMI. Blood samples were obtained at discharge for determination of N-terminal pro-brain natriuretic peptide (Nt-proBNP) and levels of miR-16, miR-27a, miR-101 and miR-150. Patients were assessed by echocardiography at 6 months follow-up and the wall motion index score (WMIS) was used as an indicator of left ventricular (LV) contractility. We assessed the added predictive value of miRNAs against a multi-parameter clinical model including Nt-proBNP.

Results

Patients with anterior AMI and elevated Nt-proBNP levels at discharge from the hospital were at high risk of subsequent impaired LV contractility (follow-up WMIS>1.2, n = 71). A combination of the 4 miRNAs (miR-16/27a/101/150) improved the prediction of LV contractility based on clinical variables (P = 0.005). Patients with low levels of miR-150 (odds ratio [95% confidence interval] 0.08 [0.01–0.48]) or miR-101 (0.19 [0.04–0.97]) and elevated levels of miR-16 (15.9 [2.63–95.91]) or miR-27a (4.18 [1.36–12.83]) were at high risk of impaired LV contractility. The 4 miRNA panel reclassified a significant proportion of patients with a net reclassification improvement of 66% (P = 0.00005) and an integrated discrimination improvement of 0.08 (P = 0.001).

Conclusion

Our results indicate that panels of miRNAs may aid in prognostication of outcome after AMI.     

Identification and Validation of Oncologic miRNA Biomarkers for Luminal A-like Breast Cancer

Introduction

Breast cancer is a common disease with distinct tumor subtypes phenotypically characterized by ER and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression, and altered miRNA expression has been demonstrated in a variety of cancer states presenting the potential for exploitation as cancer biomarkers. Blood provides an excellent medium for biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A-like (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting.

Methods

Blood samples were prospectively collected from patients with Luminal A-like breast cancer (n = 54) and controls (n = 56). RNA was extracted, reverse transcribed and subjected to microarray analysis (n = 10 Luminal A-like; n = 10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Expression of specific miRNAs was validated by RQ-PCR (n = 44 Luminal A; n = 46 Control) and potential relationships between circulating miRNA levels and clinicopathological features of breast cancer were investigated.

Results

Microarray analysis identified 76 differentially expressed miRNAs. ANN revealed 10 miRNAs for further analysis (miR-19b, miR-29a, miR-93, miR-181a, miR-182, miR-223, miR-301a, miR-423-5p, miR-486-5 and miR-652). The biomarker potential of 4 miRNAs (miR-29a, miR-181a, miR-223 and miR-652) was confirmed by RQ-PCR, with significantly reduced expression in blood of women with Luminal A-like breast tumors compared to healthy controls (p = 0.001, 0.004, 0.009 and 0.004 respectively). Binary logistic regression confirmed that combination of 3 of these miRNAs (miR-29a, miR-181a and miR-652) could reliably differentiate between cancers and controls with an AUC of 0.80.

Conclusion

This study provides insight into the underlying molecular portrait of Luminal A-like breast cancer subtype. From an initial 76 miRNAs, 4 were validated with altered expression in the blood of women with Luminal A-like breast cancer. The expression profiles of these 3 miRNAs, in combination with mammography, has potential to facilitate accurate subtype-specific breast tumor detection.     

The Clinical Significance of MiR-148a as a Predictive Biomarker in Patients with Advanced Colorectal Cancer

Aim

Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies.

Methods

We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival.

Results

Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93).

Discussion

MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.