Promising miRNAs Research Articles

MicroRNA in Human Acute Kidney Injury: A Systematic Review Protocol.

Background:Acute kidney injury (AKI) is a common complication of hospitalization with high morbidity and mortality for which no effective treatments exist and for which current diagnostic tools have limitations for earlier identification. MicroRNAs (miRNAs) are small non-coding RNAs that have been implicated in the pathogenesis of AKI, and some miRNAs have shown promise as therapeutic tools in animal models of AKI. However, less is known about the role of miRNAs in human AKI.Objective:To evaluate the role of miRNAs in human subjects with AKI.Design:Systematic review and meta-analysisMeasurements:Quantification of miRNA levels from human blood, urine, or kidney biopsy samples, and measures of renal function as defined in the study protocol.Methods:A comprehensive search strategy for Ovid MEDLINE All, Embase, Web of Science, and CENTRAL will be developed to identify investigational studies that evaluated the relationship between miRNA levels and human AKI. Primary outcomes will include measurements of kidney function and miRNA levels. Study screening, review and data extraction will be performed independently by 2 reviewers. Study quality and certainty of evidence will be assessed with validated tools. A narrative synthesis will be included and the possibility for meta-analysis will be assessed according to characteristics of clinical and statistical heterogeneity between studies.Limitations:These include (1) lack of randomized trials of miRNAs for the prevention or treatment of human AKI, (2) quality of included studies, and (3) sources of clinical and statistical heterogeneity that may affect strength and reproducibility of results.Conclusion:Previous studies of miRNAs in different animal models of AKI have generated strong interest on their use for the prevention and treatment of human AKI. This systematic review will characterize the most promising miRNAs for human research and will identify methodological constraints from miRNA research in human AKI to help inform the design of future studies.Systematic review registration:PROSPERO CRD42020201253

Extracellular Vesicles Loaded miRNAs as Potential Modulators Shared Between Glioblastoma, and Parkinson's and Alzheimer's Diseases.

Glioblastoma (GBM) is the deadliest brain tumor. Its poor prognosis is due to cell heterogeneity, invasiveness, and high vascularization that impede an efficient therapeutic approach. In the past few years, several molecular links connecting GBM to neurodegenerative diseases (NDDs) were identified at preclinical and clinical level. In particular, giving the increasing critical role that epigenetic alterations play in both GBM and NDDs, we deeply analyzed the role of miRNAs, small non-coding RNAs acting epigenetic modulators in several key biological processes. Specific miRNAs, transported by extracellular vesicles (EVs), act as intercellular communication signals in both diseases. In this way, miRNA-loaded EVs modulate GBM tumorigenesis, as they spread oncogenic signaling within brain parenchyma, and control the aggregation of neurotoxic protein (Tau, Aβ-amyloid peptide, and α-synuclein) in NDDs. In this review, we highlight the most promising miRNAs linking GBM and NDDs playing a significant pathogenic role in both diseases.

Serum miRNAs associated with tumor-promoting cytokines in non-small cell lung cancer.

Tumor-promoting cytokines are a cause of tumor progression; therefore, identifying key regulatory microRNAs (miRNAs) for controlling their production is important. The aim of this study is to identify promising miRNAs associated with tumor-promoting cytokines in non-small cell lung cancer (NSCLC). We identified circulating miRNAs from 16 published miRNA profiles. The selected miRNAs were validated in the serum of 32 NSCLC patients and compared with 33 patients with other lung diseases and 23 healthy persons using quantitative real-time PCR. The cytokine concentration was investigated using the enzyme-linked immunoassay in the same sample set, with clinical validation of the miRNAs. The correlation between miRNA expression and cytokine concentration was evaluated by Spearman's rank correlation. For consistent direction, one up-regulated miRNA (miR-145) was found in four studies, and seven miRNAs were reported in three studies. One miRNA (miR-20a) and four miRNAs (miR-25-3p, miR-223, let-7f, and miR-20b) were reported in six and five studies. However, their expression was inconsistent. In the clinical validation, serum miR-145 was significantly down-regulated, whereas serum miR-20a was significantly up-regulated in NSCLC, compared with controls. Regarding serum cytokine, all cytokines [vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and transforming growth factor β (TGF-β)], except tumor necrosis factor-α (TNF-α), had a higher level in NSCLC patients than controls. In addition, we found a moderate correlation between the TGF-β concentration and miR-20a (r = -0.537, p = 0.002) and miR-223 (r = 0.428, p = 0.015) and a weak correlation between the VEGF concentration with miR-20a (r = 0.376, p = 0.037) and miR-223 (r = -0.355, p = 0.046). MiR-145 and miR-20a are potential biomarkers for NSCLC. In addition, the regulation of tumor-promoting cytokine, through miR-20a and miR-223, might be a new therapeutic approach for lung cancer.

The Tumor Suppressive mir-148a Is Epigenetically Inactivated in Classical Hodgkin Lymphoma.

DNA methylation was shown previously to be a crucial mechanism responsible for transcriptional deregulation in the pathogenesis of classical Hodgkin lymphoma (cHL). To identify epigenetically inactivated miRNAs in cHL, we have analyzed the set of miRNAs downregulated in cHL cell lines using bisulfite pyrosequencing. We focused on miRNAs with promoter regions located within or <1000 bp from a CpG island. Most promising candidate miRNAs were further studied in primary Hodgkin and Reed-Sternberg (HRS) cells obtained by laser capture microdissection. Last, to evaluate the function of identified miRNAs, we performed a luciferase reporter assay to confirm miRNA: mRNA interactions and therefore established cHL cell lines with stable overexpression of selected miRNAs for proliferation tests. We found a significant reverse correlation between DNA methylation and expression levels of mir-339-3p, mir-148a-3p, mir-148a-5p and mir-193a-5 demonstrating epigenetic regulation of these miRNAs in cHL cell lines. Moreover, we demonstrated direct interaction between miR-148a-3p and IL15 and HOMER1 transcripts as well as between mir-148a-5p and SUB1 and SERPINH1 transcripts. Furthermore, mir-148a overexpression resulted in reduced cell proliferation in the KM-H2 cell line. In summary, we report that mir-148a is a novel tumor suppressor inactivated in cHL and that epigenetic silencing of miRNAs is a common phenomenon in cHL.

MiR-223-3p and miR-122-5p as circulating biomarkers for plaque instability.

BackgroundIn this study, we discovered and validated candidate microRNA (miRNA) biomarkers for coronary artery disease (CAD).MethodCandidate tissue-derived miRNAs from atherosclerotic plaque material in patients with stable coronary artery disease (SCAD) (n=14) and unstable coronary artery disease (UCAD) (n=25) were discovered by qPCR-based arrays. We validated differentially expressed miRNAs, along with seven promising CAD-associated miRNAs from the literature, in the serum of two large cohorts (n=395 and n=1000) of patients with SCAD and UCAD and subclinical atherosclerosis (SubA) and controls, respectively.ResultFrom plaque materials (discovery phase), miR-125b-5p and miR-193b-3p were most upregulated in SCAD, whereas miR-223-3p and miR-142-3p were most upregulated in patients with UCAD. Subsequent validation in serum from patients with UCAD, SCAD, SubA and controls demonstrated significant upregulation of miR-223-3p, miR-133a-3p, miR-146-3p and miR-155-5p. The ischaemia-related miR-499-5p was also highly upregulated in patients with UCAD compared with the other groups (SCAD OR 20.63 (95% CI 11.16 to 38.15), SubA OR 96.10 (95% CI 40.13 to 230.14) and controls OR 15.73 (95% CI 7.80 to 31.72)). However, no significant difference in miR-499-5p expression was observed across SCAD, SubA and controls. MiR-122-5p was the only miRNA to be significantly upregulated in the serum of both patients with UCAD and SCAD.ConclusionIn conclusion, miR-122-5p and miR-223-3p might be markers of plaque instability.

Identification of potential microRNA panels for pancreatic cancer diagnosis using microarray datasets and bioinformatics methods

Pancreatic cancer (PC) is a malignancy with little/no warning signs before the disease reaches its ultimate stages. Currently early detection of PC is very difficult because most patients have non-specific symptoms leading to postponing the correct diagnosis. In this study, using multiple bioinformatics tools, we integrated various serum expression profiles of miRNAs to find the most significant miRNA signatures helpful in diagnosis of PC and constructed novel miRNA diagnosis models for PC. Altogether, 27 differentially expressed miRNAs (DEMs) showed area under curve (AUC) score >80%. The most promising miRNAs, miR-1469 and miR-4530, were individually able to distinguish two groups with the highest specificity and sensitivity. By using multivariate cox regression analyses, 5 diagnostic models consisting of different combinations of miRNAs, based on their significant expression algorithms and functional properties were introduced. The correlation model consisting of miR-125a-3p, miR-5100 and miR-642b-3p was the most promising model in the diagnosis of PC patients from healthy controls with an AUC of 0.95, Sensitivity 0.98 and Specificity 0.97. Validation analysis was conducted for considered miRNAs on a final cohort consist of the microarray data from two other datasets (GSE112264 & GSE124158) . These results provide some potential biomarkers for PC diagnosis after testing in large case-control and cohort studies.

Circulating RNAs as Potential Biomarkers in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex multi-system neurodegenerative disorder with currently limited diagnostic and no therapeutic options. Despite the intense efforts no clinically applicable biomarkers for ALS are yet established. Most current research is thus focused, in particular, in identifying potential non-invasive circulating biomarkers for more rapid and accurate diagnosis and monitoring of the disease. In this review, we have focused on messenger RNA (mRNA), non-coding RNAs (lncRNAs), micro RNAs (miRNAs) and circular RNA (circRNAs) as potential biomarkers for ALS in peripheral blood serum, plasma and cells. The most promising miRNAs include miR-206, miR-133b, miR-27a, mi-338-3p, miR-183, miR-451, let-7 and miR-125b. To test clinical potential of this miRNA panel, a useful approach may be to perform such analysis on larger multi-center scale using similar experimental design. However, other types of RNAs (lncRNAs, circRNAs and mRNAs) that, together with miRNAs, represent RNA networks, have not been yet extensively studied in blood samples of patients with ALS. Additional research has to be done in order to find robust circulating biomarkers and therapeutic targets that will distinguish key RNA interactions in specific ALS-types to facilitate diagnosis, predict progression and design therapy.

Serum levels of hsa-miR-16-5p, hsa-miR-29a-3p, hsa-miR-150-5p, hsa-miR-155-5p and hsa-miR-223-3p and subsequent risk of chronic lymphocytic leukemia in the EPIC study.

Chronic lymphocytic leukemia (CLL) is an incurable disease accounting for almost one-third of leukemias in the Western world. Aberrant expression of microRNAs (miRNAs) is a well-established characteristic of CLL, and the robust nature of miRNAs makes them eminently suitable liquid biopsy biomarkers. Using a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC), the predictive values of five promising human miRNAs (hsa-miR-16-5p, hsa-miR-29a-3p, hsa-miR-150-5p, hsa-miR-155-5p and hsa-miR-223-3p), identified in a pilot study, were examined in serum of 224 CLL cases (diagnosed 3 months to 18 years after enrollment) and 224 matched controls using Taqman based assays. Conditional logistic regressions were applied to adjust for potential confounders. The median time from blood collection to CLL diagnosis was 10 years (p25-p75: 7-13 years). Overall, the upregulation of hsa-miR-150-5p, hsa-miR-155-5p and hsa-miR-29a-3p was associated with subsequent risk of CLL [OR1∆Ct-unit increase (95%CI) = 1.42 (1.18-1.72), 1.64 (1.31-2.04) and 1.75 (1.31-2.34) for hsa-miR-150-5p, hsa-miR-155-5p and hsa-miR-29a-3p, respectively] and the strongest associations were observed within 10 years of diagnosis. However, the predictive performance of these miRNAs was modest (area under the curve <0.62). hsa-miR-16-5p and hsa-miR-223-3p levels were unrelated to CLL risk. The findings of this first prospective study suggest that hsa-miR-29a, hsa-miR-150-5p and hsa-miR-155-5p were upregulated in early stages of CLL but were modest predictive biomarkers of CLL risk.

Abstract P4-10-17: Baseline and pharmacodynamic changes of circulating exosomal microRNAs predict early versus late progression to palbociclib plus endocrine therapy in patients with metastatic breast cancer. A sub-analysis of the PARSIFAL-1 trial

Abstract BACKGROUND: Palbociclib in combination with endocrine therapy (ET) is the first- or second-line standard of care for patients (pts) with hormone receptor (HR)-positive (+)/human epidermal growth factor receptor 2 (HER2)-negative (-) metastatic breast cancer (MBC). No clinically validated markers of long-term benefit from palbociclib have been established and the clinically relevant resistance mechanisms to cyclin-dependent kinases 4 and 6 inhibitors remain undefined. In the present study, we explored candidate circulating exosomal microRNAs (miRNAs) as putative predictors of benefit and/or resistance to palbociclib in combination with ET in pts included in the PARSIFAL-1 trial (ClinicalTrials.gov identifier: NCT02491983). MATERIALS AND METHODS: PARSIFAL-1 was a randomized, open-label, phase II trial aimed at evaluating the efficacy and safety of palbociclib plus either fulvestrant or letrozole in HR+/HER2- MBC pts. For the study of exosomal miRNAs, forty-five consenting pts were selected based on primary endocrine resistance according to the ABC-4 criteria. Nine pts who progressed within the first six months after treatment initiation in the absence of an objective response were considered Resistant and thirty-six pts who progressed more than six months after starting therapy were classified as Sensitive. Exosomes were isolated from plasma samples collected at study entry (baseline) and after 12 weeks of treatment initiation. Differences in miRNA expression between Resistant and Sensitive pts as well as miRNA pharmacodynamic changes between baseline and 12-week samples were assessed. Ribonucleic acid (RNA) was isolated using the miRNeasy plasma kit and the library preparation was done using the QIAseq miRNA library kit according to manufacturer’s instructions. All experiments were conducted at QIAGEN Genomic Services in Germany. Differential expression of miRNA between different conditions was studied using EdgeR statistical software package from Bioconductor. Estimated p-values for significantly differentially expressed miRNAs were adjusted using Benjamini-Hochberg’s False Discovery Rate (FDR). Differences in expression of miRNA with an FDR below 0.05 were considered significant. RESULTS: Sequencing of exosomal RNA and preparation of miRNA libraries were successful for all included samples, with good technical performance. On average, 2.8 million Unique Molecular Index-corrected reads were obtained for each sample and the average percentage of mappable reads was 34.5%. Overall, expression of miRNAs was higher in Resistant compared with Sensitive pts. Eight miRNAs were called as differentially expressed between the two groups. Four miRNAs (miR-1246; miR-375; miR-193a-5p; miR-181d-5p) were differentially expressed at baseline, three (miR-196a-5p, miR-200a-3p, miR-320d) were differentially expressed at 12 weeks, and expression of one miRNA (miR-141-3p) was consistently higher across both time points in Resistant pts. Significant pharmacodynamic changes in miRNA expression were observed both in Resistant and Sensitive pts. Seven miRNAs (miR-224-5p, miR-16-5p, let-7a-5p, miR-381-3p, miR-200c-3p, miR-493-3p, let-7b-5p) in Sensitive pts and three miRNAs (miR-223-3p, miR-126-3p, miR-320b) in Resistant pts were differentially expressed between baseline and 12-week samples. CONCLUSIONS: Circulating exosomal miRNA profiling is feasible in liquid biopsies from MBC pts. Differential expression of selected miRNAs at baseline or their pharmacodynamic modulation may predict benefit from palbociclib combined with ET in pts with HR+/HER2- MBC. Validation of the most promising miRNAs by custom quantitative PCR is warranted. Citation Format: David Casadevall, Joan Albanell Mestres, Federico Rojo, Beatríz Bellosillo, Abel González, Violeta Serra, Meritxell Bellet, Miguel Angel Gil, Miquel Angel Pujana, Angelo Gámez, Enrique Espinosa, Peter Schmid, Joseph Gligorov, Frederik Marmé, Carlos L. Arteaga, Leonardo Mina, Andrea Malfettone, Miguel Sampayo, José Manuel Pérez-García, Javier Cortés, Antonio Llombart-Cussac. Baseline and pharmacodynamic changes of circulating exosomal microRNAs predict early versus late progression to palbociclib plus endocrine therapy in patients with metastatic breast cancer. A sub-analysis of the PARSIFAL-1 trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-17.

Circulating microRNAs as promising diagnostic biomarkers for pancreatic cancer: a systematic review.

Pancreatic cancer (PC) is one of the most common forms of malignant tumors and causes of tumor-related death worldwide. The current prognosis of PC still remains poor due to the lack of effective early detection method. Recently, there is strong support that circulating miRNAs can be used as biomarkers for early detection of various cancers, including PC. The purpose of this review is to provide an overview of previous published studies on circulating miRNAs in plasma/serum for early detection of PC and summarize their diagnostic value. PubMed, Embase and Web of Science were systematically searched for eligible studies on circulating miRNAs for PC detection. Overall, 29 studies published between 2009 and 2018 evaluating 51 individual miRNAs (no P-value exceeding 0.05) and 13 miRNAs panels were included. Generally, the diagnostic performance of circulating miRNAs for PC detection was strong, with both the sensitivity and specificity of 36% individual miRNAs and 40% miRNAs panels exceeding 80%. Moreover, two promising miRNA panels were discovered and verified externally with all AUC values exceeding 0.95. Therefore, circulating miRNAs may hold potential to be used as noninvasive diagnostic biomarkers for PC, but large-scale studies are still needed to validate the promising miRNAs and optimize the miRNA panels. Since, the tremendous heterogeneity of studies in this field hampers translating miRNA markers into clinical practice, miRNA analytical procedures are also needed to be standardized in the future.

Integrating random walk and binary regression to identify novel miRNA-disease association

BackgroundIn the last few decades, cumulative experimental researches have witnessed and verified the important roles of microRNAs (miRNAs) in the development of human complex diseases. Benefitting from the rapid growth both in the availability of miRNA-related data and the development of various analysis methodologies, up until recently, some computational models have been developed to predict human disease related miRNAs, efficiently and quickly.ResultsIn this work, we proposed a computational model of Random Walk and Binary Regression-based MiRNA-Disease Association prediction (RWBRMDA). RWBRMDA extracted features for each miRNA from random walk with restart on the integrated miRNA similarity network for binary logistic regression to predict potential miRNA-disease associations. RWBRMDA obtained AUC of 0.8076 in the leave-one-out cross validation. Additionally, we carried out three different patterns of case studies on four human complex diseases. Specifically, Esophageal cancer and Prostate cancer were conducted as one kind of case study based on known miRNA-disease associations in HMDD v2.0 database. Out of the top 50 predicted miRNAs, 94 and 90% were respectively confirmed by recent experimental reports. To simulate new disease without known related miRNAs, the information of known Breast cancer related miRNAs was removed. As a result, 98% of the top 50 predicted miRNAs for Breast cancer were confirmed. Lymphoma, the verified ratio of which was 88%, was used to assess the prediction robustness of RWBRMDA based on the association records in HMDD v1.0 database.ConclusionsWe anticipated that RWBRMDA could benefit the future experimental investigations about the relation between human disease and miRNAs by generating promising and testable top-ranked miRNAs, and significantly reducing the effort and cost of identification works.

MicroRNA of whole milk samples are not suitable for pregnancy detection in cattle

Common pregnancy detection methods in dairy cattle have limits in sensitivity and timing. Thus, we aimed at finding new early pregnancy biomarkers in milk. A previous study in milk cells and skim milk suggested the possible use of several miRNAs for this purpose. In order to make the sampling procedure more convenient for daily use and to include the milk fat into analysis, here, a similar study was repeated using whole fresh milk of days 4, 12, 18 and 21 of pregnancy and non-pregnant day-matched controls, respectively. The microRNAs (miRNAs) were extracted from milk samples and sequenced on a high throughput sequencer (RNAseq). Promising miRNAs were validated via RT-qPCR. Several miRNAs were significantly regulated and six miRNAs (bta-miR-221, bta-miR-223, bta-miR-93, bta-miR-200c, bta-miR-125b and the closely related bta-miR-15b) showed a continuous trend, similar as in the milk cells of the previous study. However, pregnant and cyclic animals could not be separated completely and RT-qPCR validation of the RNAseq data was unsuccessful. Thus, miRNA of bovine whole fresh milk samples failed to show suitability for early pregnancy diagnosis.

Dynamic changes of circulating miRNAs induced by the Ebola virus vaccine VSV-EBOV

VSV-EBOV is a replication-competent Ebola virus (EBOV) vaccine, which was tested in clinical trials as response to the Ebola virus disease (EVD) outbreak 2013–2016. It is the most advanced EBOV candidate currently in the licensure process. The experimental vaccine was again administered as response to outbreaks in the Democratic Republic of Congo. However, underlying molecular mechanisms that convey protection remain incompletely understood. MicroRNAs (miRNAs) are known key regulators that influence gene expression on a post-transcriptional level. The miRNA-mediated control has emerged as a critical regulatory principle in the immune system, which strongly influences the balance of innate and adaptive immune responses by modulation of signaling pathways critical for differentiation of immune cells. We investigated expression levels of circulating miRNAs (c-miRNAs) in plasma from healthy vaccinees, as they may reflect cellular dynamics following VSV-EBOV immunization and additionally may serve as potential biomarkers for vaccine efficacy.As part of the WHO-led VEBCON consortium, we investigated safety and immunogenicity of VSV-EBOV in a phase I trial. A comprehensive analysis of expression levels on c-miRNAs from plasma samples following VSV-EBOV immunization (day 0, 1, 3 post vaccination) was conducted using RT-qPCR assays. Potential biological relevance was assessed using in silico analyses. Additionally, we correlated dynamics of miRNA expressions with our previously reported data on vaccine-induced antibody and cytokine responses and finally evaluated the prognostic power by generating ROC curves.We identified four promising miRNAs (hsa-miR-146a, hsa-miR-126, hsa-miR-199a, hsa-miR-484), showing a strong association with adaptive immune responses, exhibited favourable prognostic performance and are implicated in immunology-related functions.Our results provide evidence that miRNAs may serve as useful biomarkers for prediction of vaccine-induced immunogenicity. Furthermore, our unique data set provides insight into molecular mechanisms that underlie VSV-EBOV-mediated protective immune responses, which may help to decipher VSV-EBOV immune signature and accelerate strategic vaccine design or personalized approaches.

A comprehensive evaluation for polymorphisms in let-7 family in cancer risk and prognosis: a system review and meta-analysis

miRNA polymorphisms had potential to be biomarkers for cancer susceptibility and prognosis. The mature miRNA-let-7 family was considered as the most important miRNA for the cancer incidence and progression. Recently, the promising let-7 miRNAs were reported to be associated with various cancers, but the results were inconsistent. We performed a first-reported systematic review with a meta-analysis for the association of let-7 family single nucleotide polymorphisms (SNPs) with cancer risk/prognosis. Ten studies were included with a total of 3878 cancer cases and 4725 controls for the risk study and 1665 cancer patients for the prognosis study in this meta-analysis. In the risk study, the let-7i rs10877887 and let-7a-1/let-7f-1/let-7d rs13293512 were shown no significant association for the overall cancer risk. In the stratified analysis, the rs10877887 variant genotype was significantly associated with a decreased cancer risk in head and neck cancer (TC compared with TT: P=0.017; odds ratio (OR) = 0.81; TC + CC compared with TT: P=0.020; OR = 0.82). In the prognosis study, the let-7i rs10877887 SNP was shown to be associated with a higher risk for cancer prognosis in the dominate model (P=0.004; hazard ratio (HR) = 1.32). The other two SNPs (let-7a-1 rs10739971 and let-7a-2 rs629367) were not found to be associated with cancer survival. None of the let-7 family polymorphisms had potential to be biomarkers for cancer susceptibility but let-7i rs10877887 SNP had potential to be predicting markers for cancer prognosis. In the future, large-sample studies are still needed to verify our findings.

PO-483 Phenotype switching as an escape mechanism to targeted therapies in melanoma

IntroductionMelanoma is an aggressive skin cancer with increasing incidence worldwide. The development of BRAF kinase inhibitors as targeted treatments for patients with BRAF-mutant tumours and the introduction of immunotherapies contributed profoundly to an improved overall survival of patients with metastatic melanoma. Despite these promising results, the emergence of rapid resistance to these therapeutic approaches remains a serious clinical issue.Material and methodsTo investigate the impact of BRAF inhibitors on miRNomes and transcriptomes, we used in vitro melanoma models consisting of BRAF inhibitor-sensitive and -resistant cell lines generated in our laboratory. Subsequently, miRNA and gene expression analyses were performed in order to identify the underlying mechanisms of resistance.Results and discussionsRegarding miRNome and transcriptome changes, the long-term effects of BRAF inhibition differed in a cell line-specific manner with the two different BRAF inhibitors inducing comparable responses in drug-sensitive melanoma cell lines. Despite this heterogeneity, several miRNAs (e.g. miR-100–5 p) and genes (e.g. AXL) were distinctly differentially expressed in drug-resistant versus -sensitive cell lines. Analyses of co-expressed miRNAs, as well as inversely correlated miRNA-mRNA pairs, revealed a switch from a MITFhigh to an AXLhigh ratio in a subset of drug-resistant melanoma cell lines that might be regulated by miRNAs. Additionally, the inhibition of AXL reduces growth of BRAF inhibitor-resistant melanoma cells, thus the combined inhibition of BRAF and AXL might be beneficial for patients with metastatic melanoma.ConclusionIn this study, promising miRNAs and genes were identified and associated to BRAF inhibitor-mediated resistance in melanoma, and might be considered as prognostic and/or diagnostic resistance biomarkers in melanoma drug resistance.

Vernalization can regulate flowering time through microRNA mechanism in Brassica rapa.

Vernalization is an important process that regulates the floral transition in plants. MicroRNAs (miRNAs) are endogenous non-coding small RNA (sRNA) molecules that function in plant growth and development. Despite that miRNAs related to flowering have previously been characterized, their roles in response to vernalization in pak-choi (Brassica rapa ssp. chinensis) has never been studied. Here, two sRNA libraries from B. rapa leaves (vernalized and non-vernalized plants) were constructed and sequenced. Two hundred eight known and 535 novel miRNAs were obtained, of which 20 known and 66 new miRNAs were significantly differentially expressed and considered as vernalization-related miRNAs. The corresponding targets were predicted on the basic of sequence homology search. In addition, 11 miRNAs and eight targets were selected for real-time quantitative PCR to confirm their expression profiles. Functional annotation of targets using gene ontology and Kyoto encyclopedia of genes and genomes results suggested that most targets were significantly enriched in the hormone signaling pathway. Moreover, a decreased indole-3-acetic acid (IAA) and an increased GA3 hormone were detected after vernalization, indicating that the IAA and GA3 might response to vernalization. These results indicated that vernalization regulates flowering through microRNA mechanism by affecting endogenous hormone level in B. rapa. This study provides useful insights of promising miRNAs candidates involved in vernalization in B. rapa, and facilitates further investigation of the miRNA-mediated molecular mechanisms of vernalization in B. rapa.

MicroRNA Signature of Traumatic Brain Injury: From the Biomarker Discovery to the Point-of-Care.

Traumatic brain injury (TBI) is a serious problem that causes high morbidity and mortality around the world. Currently, no reliable biomarkers are used to assess the severity and predict the recovery. Many protein biomarkers were extensively studied for diagnosis and prognosis of different TBI severities such as S-100β, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), neurofilament light chain (NFL), cleaved tau protein (C-tau), and ubiquitin C-terminal hydrolase-L1 (UCH-L1). However, none of these candidates is currently used in the clinical practice, due to relatively low sensitivity, for the diagnosis of mild TBI (mTBI) or mild to moderate TBI (MMTBI) patients who are clinically well and do not have a detectable intracranial pathology on the scans. MicroRNAs (miRNAs or miRs) are a class of small endogenous molecular regulators, which showed to be altered in different pathologies, including TBI and for this reason, their potential role in diagnosis, prognosis and therapeutic applications, is explored. Promising miRNAs such as miR-21, miR-16 or let-7i were identified as suitable candidate biomarkers for TBI and can differentiate mild from severe TBI. Also, they might represent new potential therapeutic targets. Identification of miRNA signature in tissue or biofluids, for several pathological conditions, is now possible thanks to the introduction of new high-throughput technologies such as microarray platform, Nanostring technologies or Next Generation Sequencing. This review has the aim to describe the role of microRNA in TBI and to explore the most commonly used techniques to identify microRNA profile. Understanding the strengths and limitations of the different methods can aid in the practical use of miRNA profiling for diverse clinical applications, including the development of a point-of-care device.

Abstract P1-07-31: Integrated transcriptomics analyses identify novel three microRNAs signature to predict poor prognosis and metastasis in breast cancer

Abstract Backgrounds: MicroRNA (miRNA) play a crucial role in cancer progression, and altered miRNA expression has been demonstrated to be associated with breast cancer. MiRNA is also known to be stably detectable not only in tissue but peripheral circulation; therefore it could be a feasible and potential biomarker of breast cancer in clinical settings. However, few studies have been reported to identify promising miRNA profiles as predictive biomarker using statistically satisfied large cohorts of breast cancer patients. The aim of this study is to identify miRNA signature that can predict patient survival utilizing integrated and unbiased transcriptomics analyses. Methods: Integrated and unbiased transcriptomics approach was conducted on genomic and clinicopathological information of 2580 breast cancer patients. We utilized The cancer Genome Atlas (TCGA) to identify miRNA signature that could significantly associated with clinical relevance including prognosis and metastatic information, followed by validation analyses with the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and Gene Expression Omnibus (GEO) Results: A novel risk scoring model including three miRNAs signature (miR-19a, miR-93, and miR-106a) was identified using Cox model. This miRNA score was able to extract the patient population with extremely poor prognosis in TCGA (5-yr overall survival rate, 49.2 %, p=0.0005). This result was validated with another three completely independent cohorts with microarray dataset accompanied with sufficient clinical information and miRNA expression (GSE19536, n=96, p=0.0009; GSE22220, n=210, p=0.0003; METABRIC, n=1223, p=0,0023). Interestingly, the subtype stratification with PAM-50 classification using bioinformatics pipeline demonstrated that this miRNA score could predict poor overall or disease-free survival especially in the population with luminal A, B or normal like subtype (p=0.0300 and p=0.0001, respectively). In addition, competing risk analysis for tumor recurrences demonstrated that the risk scoring using three miRNAs signature could be significantly associated with bone metastasis (p=0.0052). Finally, Gene Set Enrichment Analysis (GSEA) identified that high risk score using three miRNAs associated significantly with several critical gene sets related to metastatic formation such as angiogenesis (p &amp;lt;0.0001), epithelial mesenchymal transition (EMT) (p = 0.0155), focal adhesion (p &amp;lt;0.0001), TGF-beta signaling pathway (p = 0.0025), and ECM receptor interaction (p = 0.0068). Conclusions: We demonstrated a promising miRNAs signature score system for predicting extremely poor prognosis and metastatic potentiality in breast cancer using novel integrated transcriptomics concept. Citation Format: Takabe K, Kawaguchi T, Yan L, Qi Q, Peng X, Young J, Liu S. Integrated transcriptomics analyses identify novel three microRNAs signature to predict poor prognosis and metastasis in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-31.

Omeprazole Inhibits Cell Proliferation and Induces G0/G1 Cell Cycle Arrest through Up-regulating miR-203a-3p Expression in Barrett's Esophagus Cells.

Existing data suggest that proton pump inhibitors (PPIs), particularly omeprazole, have significant anti-tumor action in monotherapy and or combination chemotherapy. Hedgehog (Hh) signaling pathway represents a leading candidate as a molecular mediator of Barrett’s esophagus (BE). Studies have indicated reduced miRNAs in BE progression, however, little is known about the latent anti-neoplasm effects of miRNAs in BE cells. Here, we investigated whether omeprazole could inhibit BE progression by regulating Hh pathway and explored the promising Hh-targeted miRNAs in BE cells. We conducted qRT-PCR and immunoblotting assay to evaluate the effects of omeprazole on the expression of Hh signaling components and miR-203a-3p in CP-A and CP-B cells. The promising target genes of miR-203a-3p were predicted by bioinformatics methods, and verified by luciferase assays and qRT-PCR. The effects of omeprazole on BE cell proliferation and cell cycle distribution were determined. The overexpression or silencing of miR-203a-3p was performed to test its anti-proliferative effects. Finally, rescue experiments that miR-203a-3p inhibitor alleviated the effects of omeprazole on decreasing the levels of Gli1 mRNA, protein and luciferase were performed. Mechanistic studies showed that omeprazole could inhibit the expression of Gli1 and the nuclear localization of Gli1. Moreover, we determined that omeprazole could selectively up-regulated the expression of miR-203a-3p, and Gli1 was a bona fide target of miR-203a-3p. miR-203a-3p inhibitor alleviated the suppressing effects of omeprazole on Gli1 luciferase activity, mRNA and protein level. The functional assay suggested that omeprazole could dose-dependently inhibit BE cell growth and induce cell cycle arrest in G0/G1 phase. Additionally, overexpression and silencing of miR-203a-3p in BE cells disrupted cell cycle progress, resulting in suppressing and accelerating cell proliferation, respectively. Taken together, these data provide a novel mechanism of potentially anti-neoplastic effects for omeprazole through modulation of miR-203a-3p expression and thus suppressing Hh/Gli1 signaling in BE cells.

Glomerulocapillary miRNA response to HLA-class I antibody in vitro and in vivo

Changes in miRNA expression of glomerular capillaries during antibody-mediated rejection (ABMR) are poorly understood and could contribute to the deleterious inflammation and fibrosis of ABMR via suppression of target genes. A better understanding could lead to novel diagnostic tools and reveal novel therapeutic targets. We explored deregulated miRNAs in an glomeruloendothelial in vitro model of ABMR due to class I human leukocyte antigen (HLA) with and without complement activation. We studied a set of 16 promising candidate miRNAs in microdissected glomeruli a confirmation set of 20 human transplant biopsies (DSA+) compared to 10 matched controls without evidence for ABMR. Twelve out of these 16 glomerulocapillary miRNAs could successfully be confirmed as dysregulated in vivo with 10 upregulated (let-7c-5p, miR-28-3p, miR-30d-5p, miR-99b-5p, miR-125a-5p, miR-195-5p, miR-374b-3p, miR-484, miR-501-3p, miR-520e) and 2 downregulated (miR29b-3p, miR-885-5p) in DSA+ vs. controls. A random forest analysis based on glomerular miRNAs identified 18/20 DSA+ and 8/10 controls correctly. This glomerulocapillary miRNA signature associated with HLA class I-DSA could improve our understanding of ABMR and be useful for diagnostic or therapeutic purposes.