Identification Of microRNA Signatures Research Articles

Identification of microRNA Signatures in Peripheral Blood of Young Women as Potential Biomarkers for Metal Allergy

MicroRNA (miRNA) is a short (19-24 nucleotide) endogenous non-protein RNA that exists in the body and controls the translation process from genes to proteins. It has become useful as a diagnostic tool and a potential treatment target in cancer research. To explore the function of miRNA in contact dermatitis, female participants with a positive metal allergy diagnosis (n = 3) were enrolled along with additional female participants with no medical history of metal allergy (n = 3). A patch test was performed on each participant. Peripheral blood was collected from all the participants before the patch test and at days 3 and 7 after starting the patch test. After total RNA was obtained from peripheral blood leukocytes and cDNA was generated, microarray analysis was performed to analyze the large-scale circulating miRNA profile. Real-time polymerase chain reaction (RT-PCR) was then used to clarify the overall target miRNA expression. Downregulation of hsa-let-7d-5p, hsa-miR-24-3p, hsa-miR-23b-3p, hsa-miR-26b-5p, and hsa-miR-150-5p was found on day 7. Certain miRNAs were confirmed using RT-PCR. These peripheral blood miRNAs could be diagnostic biomarkers for metal allergies.

Identification of microRNA Signature and Key Genes Between Adenoma and Adenocarcinomas Using Bioinformatics Analysis.

BackgroundIn worldwide, colorectal cancer (CRC) is very common and the mechanisms remain unclear. This study aims to identify between adenomas with epithelial dislocation (false invasion) and adenomas with early adenocarcinoma (true invasion).MethodsGSE41655 and GSE57965 datasets were obtained in the Gene Expression Omnibus (GEO) database. microRNA expression profiles and clinicopathological data from the TCGA (The Cancer Genome Atlas) database were downloaded to further validate the results in GEO. GEO software and the GEO2R calculation method were used to analyze two gene profiles. The co-expression of differentially expressed microRNAs (DEMs) and genes (DEGs) were identified and searched in the FunRich databases for pathway and ontology analysis. Cytoscape was utilized to construct the mRNA-microRNA network. Validation of gene expression levels was conducted by online databases and qRT-PCR and IHC experiments.ResultsIn total, 6 DEMs and 34 DEGs are selected after calculating. KEGG results indicated that genes are enriched in certain tumor associated pathways. Four out of 6 microRNAs had a significant relationship with the overall survival (P < 0.05) and showed a good performance in predicting the survival risk of patients with colorectal carcinoma. Furthermore, expression levels of hsa-miR-455 and hsa-miR-125a were then verified by qRT-PCR which all target BCL2L12. IHC results showed that the expression level of BCL2L12 was higher in adenocarcinoma than in adenoma. Based on the selected gene, the top 10 small molecules were screened out as potential drugs.ConclusionBy using microarray and bioinformatics analyses, DEMs and DEGs were selected and a complete gene network was constructed. To our knowledge, BCL2L12 and related molecules including hsa-miR-455 and hsa-miR-125a were firstly identified as potential biomarkers in the progression from adenoma to adenocarcinoma.

P4154MicroRNA Signatures Predict Early Major Coronary Events in Middle Aged Men and Women

Abstract Background The role of microRNA as biomarkers able to predict major coronary events (MACE) has not been fully elucidated, reproducibility being a critical issue. Aim To identify circulating microRNA signatures able to predict MACE. Methods We employed a PCR-based method to screen 754 microRNAs in a cohort of 60-year-olds (60YOs) from Stockholm, using a nested case-control design (100 cases vs 100 matched controls). The association of microRNAs and their interaction with the risk of MACE (myocardial infarction (MI), angina and sudden cardiac death) was estimated with random-effect logistic regression and expressed as OR with 95% CI. A bioinformatics approach identified microRNA clusters based on predicted targets. Main findings were tested in 58 MI and 60 age and sex matched referents from the the Nord-Trøndelag Health (HUNT) Study, a longitudinal population health study conducted in Norway. Results Fifty-five microRNAs were found to be associated with risk of MACE in the 60YO. MicroRNA-145-3p was associated with the largest estimated risk increase of MACE after adjustment for the common CV risk factors (OR: 2.18; 95% CI: 1.27–3.75). Interaction analysis revealed that increasing plasma levels of microRNA-320b modulated the association of 16 microRNAs with risk of MACE. As an example the estimated MACE risk associated with microRNA-145-3p was 1.47 (0.87–2.47) in the presence of low (&lt;25th percentile) and 4.00 (1.79- 8.93) in the presence of high (&gt;75th percentile) miRNA 320b expression levels. Sixteen microRNA pairs could be classified in 4 functional clusters with 492 predicted gene targets, mainly involved in the regulation of inflammation, thrombosis and lipid metabolism. Eight miRNAs interacting pairs belonging to cluster 2 and 4 showed a similar association trend with MI risk in the HUNT study. Conclusions We report the identification of microRNA signatures predicting risk of MACE in middle-aged Scandinavian men and women. These signatures could be a valuable tool to improve CV disease prediction in the aged.

Identification of microRNA signatures in umbilical cord blood associated with maternal characteristics.

BackgroundUmbilical cord blood could serve as useful source of blood markers enabling more efficient and reliable prenatal and neonatal diagnostics. MicroRNAs (miRNAs) are ubiquitous in body fluids where they were used for detecting and monitoring various physiological and pathological conditions. In this descriptive study, we aimed to identify changes in miRNA expression profiles associated with basic maternal somatic and epidemiological characteristics.MethodsStudy is based on 24 mothers from the Pilot phase of CELSPAC: TNG (Central European Longitudinal Studies of Parents and Children: The Next Generation) study. Cord blood was collected at time of delivery and global miRNA profiling was performed using microRNA Ready-to-use PCR Human Panel I+II TaqMan microarrays. Expression profiles were statistically evaluated in relation to maternal age, BMI, pregnancy weight gain, blood type, Rh factor status, allergies during pregnancy, addictive substance abuse and smoking status.ResultsWe analyzed expression of 752 human mature miRNAs in 24 samples of umbilical cord blood. For all maternal characteristics tested we described a specific signature of significantly deregulated miRNAs (P < 0.05). Analysis revealed seven miRNA associated with maternal age (three increased and four decreased in women younger than 35 years), 14 miRNAs associated with BMI status (five miRNAs increased and nine miRNAs decreased in women with BMI > 25) and nine miRNAs associated with maternal weight gain during pregnancy (eight miRNAs increased, and one miRNA decreased in women with weight gain < 12 kg). Additionally, 17 miRNAs correlated to blood type (two miRNAs decreased in blood type A, 11 increased in blood type B, two miRNAs increased in blood type AB and two miRNAs increased in blood type 0) and 17 miRNAs to Rh status of mother. We also detected seven miRNAs deregulated in umbilical cord blood of women with allergy (four increased and three decreased in women with allergy), four miRNAs associated to addictive substance abuse status (two up- and two downregulated in women with addictive substance abuse) and eight miRNAs associated with maternal cigarette smoking during pregnancy.ConclusionsWe successfully described differences in miRNA profiles in umbilical cord blood associated with basic characteristics connected with mother. Our data suggest that miRNAs in umbilical cord blood are detectable and associated with a wide range of maternal characteristics. These results indicate that miRNAs could potentially serve, and should be studied, as biomarkers for screening and diagnosis of pregnancy-associated complications and pathologies.

Circulating microRNA signatures identified in early versus late knee osteoarthritis

Purpose: There continues to be a lack of specific osteoarthritis (OA) biomarkers that can be used in a therapeutic, prognostic, or diagnostic manner. Since microRNAs are promising biomarkers for several diseases, we previously used microRNA PCR-arrays and identified a panel of microRNAs in the synovial fluid that differentiate between early- and late-stage radiographic knee OA. We also performed microRNA microarrays and identified 2 microRNAs as mediators of cartilage degeneration. However, the gold standard approach to identifying biomarkers is next generation sequencing because it offers the sensitivity and specificity to detect novel and low abundance microRNAs that are unique to various cohorts (early versus late OA, men versus women, young versus old, normal weight versus overweight). Here, we use next generation sequencing to identify signatures of circulating microRNAs as biomarkers for knee OA. We hypothesize that identification and characterization of known and novel circulating microRNAs can be used as biomarker signatures to define cohorts of patients with OA. Methods: Plasma samples from knee OA patients are obtained from the Knee Osteoarthritis BioBank at the University Health Network in Toronto, Canada. Samples include healthy donors with no history of musculoskeletal disease (N=100), Kellgren-Lawrence grades 0 and 1 for early OA (N=100), and Kellgren-Lawrence grades 3 and 4 for late OA (N=750). Patients with comorbidities that might affect microRNA signatures are excluded. In a pilot experiment, plasma samples from 5 healthy donors, 5 early OA patients, and 5 late OA patients were subjected to next generation sequencing of microRNAs. Differentially expressed microRNAs were identified using the negative binomial exact test. To validate our findings, we are currently sequencing plasma from larger cohorts of patients. Further statistical analysis will be used to identify relationships between microRNA signatures and known risk factors such as sex, age, and body mass index. Bioinformatic methods will be used to predict the targets and biological function of candidate microRNAs in OA. Results: After mapping sequencing reads to mirbase20, 164 microRNAs with greater than 10 counts per million were identified in all samples. Principal component analysis was performed using the 50 microRNAs with the largest coefficient of variation based on normalized counts. This revealed a clear separation of early OA samples from both late OA and healthy samples. The most differentially expressed microRNAs were identified based on false discovery rate less than 0.05, log counts per million greater than 2, and log fold change greater than 1.5. Hierarchical clustering of these microRNAs revealed a distinct pattern where 60 microRNAs were upregulated only in early OA samples (Figure 1). Among these are 4 novel putative microRNAs that have not previously been identified. We are currently validating these findings with additional sequencing experiments in each group (N=100 healthy donors, N=100 early OA, N=750 late OA) to establish a clear microRNA signature for early knee OA. Conclusions: Data from our pilot experiment demonstrate that next generation sequencing is a useful approach for identifying known and novel circulating microRNAs in OA. Sequencing of early OA samples will allow identification of microRNA signatures that can be used to distinguish these patients from late OA patients. Circulating microRNAs may represent valid and reliable biomarkers with potential applications for improving OA detection and treatment.

Genome-wide identification of microRNA signatures associated with stem/progenitor cells in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a malignant transformation with uncontrolled proliferation of lymphoid precursor cells within bone marrow including a dismal prognosis after relapse. Survival of a population of quiescent leukemia stem cells (LSCs, also termed leukemia-initiating cells (LICs)) after treatment is one of the relapse reasons in Ph+ ALL patient. MicroRNAs (miRNAs) are known as highly conserved 19-24 nucleotides non-protein-coding small RNAs that regulate the expression of human genes. miRNAs are often involved in the tuning of hematopoiesis. Therefore, the deregulation of miRNA expression and function in hematopoietic cells can cause cancer and promote its progression. This is the first comprehensive analysis of miRNA expression differences between CD34+CD38- LSCs and CD34+CD38+ leukemic progenitors (LPs) from the same Ph+ B-ALL bone marrow samples using high-throughput sequencing technologies. We identified multiple differentially expressed miRNAs including hsa-miR-3143, hsa-miR-6503-3p, hsa-miR-744-3p, hsa-miR-1226-3p, hsa-miR-10a-5p, hsa-miR-4658 and hsa-miR-493-3p related to LSC and LP populations which have regulatory functions in stem-cell associated biological processes. The deregulation of these miRNAs could affect leukemogenesis, clonogenic and stemness capacities in these subpopulations of Ph+ B-ALL. Therefore, identification of these LSC associated miRNAs may improve the diagnosis and management of B-ALL. These findings may also lead to future strategies to eliminate the presence of resistant LSCs, either by induction of apoptosis or by sensitizing these cells to chemotherapy.

Next generation sequencing discovers known and novel circulating micrornas in knee osteoarthritis

Purpose: There are no biomarkers that can be used in a therapeutic, prognostic, or diagnostic manner for osteoarthritis (OA). Circulating microRNAs have emerged as promising biomarkers for a variety of diseases. MicroRNAs are small non-coding RNAs that repress expression of target genes. Our lab was the first to perform global array screening of microRNAs in OA, but this approach identifies only known microRNAs. The aim of this study is to use next generation sequencing to identify novel circulating microRNAs in patients with OA. This technology has never been applied to identifying circulating microRNAs in OA, and has the sensitivity and specificity to detect microRNAs that are unique to various cohorts, including early OA versus late OA, men versus women, young versus old, and normal weight versus overweight. These data will be used to establish biomarker signatures which distinguish cohorts of OA patients. Methods: Plasma samples from OA patients are obtained from the Knee Osteoarthritis BioBank at the University Health Network in Toronto, Canada. Cohorts are defined based on Kellgren-Lawrence radiographic grading for early OA (grades 0 & 1) and late OA (grades 3 & 4). Patients with comorbidities that might affect microRNA signatures are excluded. Plasma samples from 10 normal donors, 10 early OA patients, and 10 late OA patients will be subjected to next generation sequencing of microRNAs. Data analysis will be performed in R 3.2.2 using the edgeR package (v3.18.1). Candidate microRNAs will be selected for validation by real-time PCR in additional cohorts of early OA (N=100), late OA (N=100), and normal donors (N=100). Biostatistic methods will be used to identify relationships between microRNA signatures and known risk factors such as sex, age, and body mass index. Bioinformatic methods will be used to predict the targets and biological function of candidate microRNAs in OA. Results: Initial sequencing analysis of 5 normal donors and 5 late OA patients is complete. A total of 2579 known and 59 novel microRNAs were identified. These were filtered for microRNAs with at least 10 counts per million of classified sequences in at least 2 of 10 samples, resulting in 314 known and 15 novel microRNAs. From this, a list of top 20 differentially expressed microRNAs was generated based on false discovery rate < 0.05, log counts per million > 2, log fold change > 1.5, and p-value < 0.0003. Hierarchical clustering of these microRNAs revealed a distinct pattern between normal and late OA samples. Among these microRNAs is a candidate previously shown to be dysregulated in OA synovial fluid, and a novel putative microRNA that has not previously been identified. Validation of these findings by real-time PCR and additional sequencing experiments are currently underway. Conclusions: Interim results suggest that next generation sequencing is a useful approach for identifying known and novel microRNAs in OA. Sequencing of early OA samples will allow identification of microRNA signatures that can be used to distinguish these patients from late OA patients. Circulating microRNAs may represent valid and reliable biomarkers with potential applications for improving OA detection and treatment.

Identification of microRNA signature in the progression of gestational trophoblastic disease

Gestational trophoblastic disease (GTD) encompasses a range of trophoblast-derived disorders. The most common type of GTD is hydatidiform mole (HM). Some of HMs can further develop into malignant gestational trophoblastic neoplasia (GTN). Aberrant expression of microRNA (miRNA) is widely reported to be involved in the initiation and progression of cancers. MiRNA expression profile also has been proved to be the useful signature for diagnosis, staging, prognosis, and response to chemotherapy. Till now, the profile of miRNA in the progression of GTD has not been determined. In this study, a total of 34 GTN and 60 complete HMs (CHM) trophoblastic tissues were collected. By miRNA array screening and qRT-PCR validating, six miRNAs, including miR-370-3p, -371a-5p, -518a-3p, -519d-3p, -520a-3p, and -934, were identified to be differentially expressed in GTN vs. CHM. Functional analyses further proved that miR-371a-5p and miR-518a-3p promoted proliferation, migration, and invasion of choriocarcinoma cells. Moreover, we demonstrated that miR-371a-5p was negatively related to protein levels of its predictive target genes BCCIP, SOX2, and BNIP3L, while miR-518a-3p was negatively related to MST1 and EFNA4. For the first time, we proved that miR-371a-5p and miR-518a-3p directly targeted to 3′-UTR regions of BCCIP and MST1, respectively. Additionally, we found that miR-371a-5p and miR-518a-3p regulated diverse pathways related to tumorigenesis and metastasis in choriocarcinoma cells. The results presented here may offer new clues to the progression of GTD and may provide diagnostic biomarkers for GTN.

Identification of microRNA signature in different pediatric brain tumors.

Understanding pediatric brain tumor biology is essential to help on disease stratification, and to find novel markers for early diagnosis. MicroRNA (miRNA) expression has been linked to clinical outcomes and tumor biology. Here, we aimed to detect the expression of different miRNAs in different pediatric brain tumor subtypes to discover biomarkers for early detection and develop novel therapies. Expression of 82 miRNAs was detected in 120 pediatric brain tumors from fixed-formalin paraffin-embedded tissues, low-grade glioma, high-grade glioma, ependymoma, and medulloblastoma, using quantitative real-time PCR. Low-expression of miR-221, miR-9, and miR-181c/d and over-expression of miR-101, miR-222, miR-139, miR-1827, and miR-34c was found in medulloblastoma; low expression of miR-10a and over-expression of miR-10b and miR-29a in ependymoma; low expression of miR-26a and overexpression of miR-19a/b, miR-24, miR-27a, miR- 584, and miR-527 in low-grade glioma. Cox regression showed differential miRNA expression between responders and non-responders. The most specific were miR-10a and miR-29a low expression in LGG non-responders, miR-135a and miR-146b over-expression in ependymoma non-responders, and miR-135b overexpression in medulloblastoma non-responders. MicroRNAs are differentially expressed in subtypes of brain tumors suggesting that they may help diagnosis. A greater understanding of aberrant miRNA in pediatric brain tumors may support development of novel therapies.

Identification of microRNA signature and potential pathway targets in prostate cancer.

Prostate cancer (PC) is the most common and the second leading cause of cancer-related death among American men. Early diagnosis is a prerequisite to improving therapeutic benefits. However, the current clinical biomarkers for PC do not reliably decipher indolent PC from other urogenital disorders. Thus, effective clinical intervention necessitates development of new biomarkers for early detection of PC. The present study aimed to identify the miRNA signature in organ-confined (Gleason Score 6) prostate tumors. MicroRNA (miRNA/miR) array analysis identified 118 upregulated and 73 downregulated miRNAs in microdissected tumors in comparison to matched neighboring normal prostate epithelium. The miRs-Plus-A1083, -92b-5p, -18a-3p, -19a-3p, -639, -3622b-3p, -3189-3p, -155-3p, -410, -1179, 548b-5p, and -4469 are predominantly expressed (7-11-fold), whereas miRs-595, 4490, -3120-5p, -1299, -21-5p, -3677-3, -let-7b-5p, -5189, 3-121-5p, -4518, -200a-5p, -3682-5p, -3689d, -3149 represent the most downregulated (12-113-fold) miRNAs in microdissected prostate tumors. The array expression profile of selected miRNA signature and their potential mRNA targets was validated by qRT-PCR analysis in PC cell lines. Integrated in silico and computational prediction analyses demonstrated that the dysregulated miRNA signature map to key regulatory factors involved in tumorigenesis, including cell cycle, apoptosis, and p53 pathways. The newly identified miRNA signature has potential clinical utility as biomarkers, prognostic indicators, and therapeutic targets for early detection of PC. Further studies are needed to assess the functional significance and clinical usefulness of the identified miRNAs. Impact Statement To our knowledge his is the first study of identifying miRNA signatures in microdissected indolent (Gleason score 6) prostate cancer in comparison to matched normal prostate epithelium. By employing in silico and computational prediction analysis, the study provides a landscape of potential miRNA targets and key cellular pathways involved in prostate tumorigenesis. Identification if miRNAs and their relevant targets and pathways pave the way for underpinning their mechanistic role of miRNAs in human prostate tumorigenesis, and possibly other human cancers. Importantly, the outcome of the study has important clinical implications for the management of prostate cancer, including the use of miRNA(s) as biomarkers for early detection of prostate cancer.