miRNAs In Samples Research Articles

MicroRNA dysregulation in ataxia telangiectasia.

Ataxia telangiectasia (AT) is a rare disorder characterized by neurodegeneration, combined immunodeficiency, a predisposition to malignancies, and high clinical variability. Profiling of microRNAs (miRNAs) may offer insights into the underlying mechanisms of complex rare human diseases, as miRNAs play a role in various biological functions including proliferation, differentiation, and DNA repair. In this study, we investigate the differential expression of miRNAs in samples from AT patients to identify miRNA patterns and analyze how these patterns are related to the disease. We enrolled 20 AT patients (mean age 17.7 ± 9.6 years old) and collected clinical and genetic data. We performed short non-coding RNA-seq analysis on peripheral blood mononuclear cells (PBMCs) and fibroblasts to compare the miRNA expression profile between AT patients and controls. We observed 42 differentially expressed (DE)-miRNAs in blood samples and 26 in fibroblast samples. Among these, three DE-miRNAs, miR-342-3p, miR-30a-5p, and miR-195-5p, were further validated in additional AT samples, confirming their dysregulation. We identified an AT-related miRNA signature in blood cells and fibroblast samples collected from a group of AT patients. We also predicted several dysregulated pathways, primarily related to cancer, immune system control, or inflammatory processes. The findings suggest that miRNAs may provide insights into the pathophysiology and tumorigenesis of AT and have the potential to serve as useful biomarkers in cancer research.

CRISPR Based Gene Editing Technologies in Cancer Research and Detection via miRNA

The CRISPR system of genetic editing has been a breakthrough in all fields of science and medicine, including that of cancer research, therapy, and diagnosis. In the last two years, research has narrowed down on CRISPRs ability to serve as a tool to detect oncogenes in patients to diagnose cancer in a quick and efficient manner. CRISPR and the three key enzymes (cas9, cas12, and cas13) are a revolutionary tool for medicine and cancer trials. MicroRNAs (miRNAs) are short noncoding RNA which regulate gene expression and gene functions, both crucial to cell functions. However, aberrant miRNA is expressed in all tumor proliferation diseases, including cancer. Studies have shown CRISPRs ability to detect cancerous levels of oncogenic MicroRNAs in human samples through a variety of methods such as CRISPR-Cas9/RNAi, CRISPR/Cas12 and RCA coupling, CRISPR/Cas13 exosome coupling, and Cas13a-Cas12a amplification. All of these methods provide extremely quick high sensitivity and high specificity detection of oncogenic miRNA in samples with concentrations below 1 fM. This paper is a synthesis of the current research on the methods mentioned above to aid in the further research of CRISPR/Cas systems in cancer diagnosis and cancer therapeutics.

High Throughput Sequencing Analysis of Exosomal miRNA Expressions in Early-Onset Preeclampsia Patients.

The goal of this study was to screen for differentially expressed exosomal miRNAs in peripheral blood samples of early-onset preeclampsia and normal pregnant patients using high-throughput sequencing methods and explore their effects on the pathogenesis of preeclampsia. Peripheral blood samples from 5 patients with early-onset preeclampsia and 5 normal pregnant women (control group) were enrolled. Then, exosomes were extracted from each sample, and the procedure was replicated three times. An Illumina HiSeq4000 sequencing platform was used to analyze exosomal miRNAs in all samples before comparison. The target genes and signaling pathway predictions and the biological function enrichments of significant and differentially expressed miRNAs were assessed using Miranda and Starbase software, as well as GO and KEGG databases. Compared with the control, patients in the early-onset preeclampsia group had 65 significantly and differentially expressed exosomal miRNAs in their peripheral blood samples. The results have shown that of the 65 differentially expressed miRNAs, 17, including has-miR-6855, has-miR-7151, and has-miR-6777, were up-regulated, and 48, including has-miR-1247, has-miR-29B2, and has-miR-941, were down-regulated (p < 0.05). The Miranda and TargetScanS algorithms predicted a total of 2,231 target genes from the differentially expressed miRNAs. The Go and KEGG analyses showed that the principal biological function of these target genes was the regulation of Ras protein signal transduction, histone modification, GTPase-mediated signal transduction, and transforming growth factor (TGF)-β. Additionally, the results also showed that the major pathways involved in the regulation of these functions were the PI3K Akt, MAPK, tumor necrosis factor, and EGFR tyrosine kinase inhibition signaling pathways. There are significant differences in the expression profiles of exosomal miRNAs between early-onset preeclampsia patients and normal pregnant women. These differentially expressed miRNAs may not only play an important regulatory role in the occurrence of early-onset preeclampsia but also participate in its pathophysiological process through genetic regulation of a variety of biological functions and signal pathways.

Study the relationship between polymorphisms of pre-miRNA regions and expression of mature miRNA in samples of Iraqi infertile men

Polymorphisms that increase risk factor for male infertility is one of the most studied infertility genetic factor however polymorphisms within pre- miRNA sequences have not extensively documented. This study aimed to highlighted association between idiopathic male infertility and, hsa-mir-196a-2 rs11614913Polymorphism genotyping and expression level of mature miRNA were done in semen samples of Iraqi fertile and infertile male in order to study impact of polymorphism on expression level of mature miRNA andstudy correlationbetween both of polymorphism genotype and mature miRNA expression level with quality of semen parameters. Results of this study demonstrated that homozygous mutant (TT) and heterozygous mutant (CT) genotypes of pre-miRNA regions (hsa-miRNA-196a-2, rs11614913) were significantly (P ≤ 0.01) higher in patients group when compared with control and associated with idiopathic Asthenozoospermia, Oligoasthenozoospermia and specifically Azoospermia.Correlation between polymorphism genotype and semen parameters exhibits that genotype obviously impacts semen parameters. Analyzing correlation between polymorphism genotype and expression elucidated that mutant homozygous (TT) genotype increasing expression level of mature miRNA. Elevated level of miRNA-196a-5p exhibitednegative correlation with semen parameter.

The Impact of the Anticoagulant Type in Blood Collection Tubes on Circulating Extracellular Plasma MicroRNA Profiles Revealed by Small RNA Sequencing.

Pre-analytical factors have a significant influence on circulating microRNA (miRNA) profiling. The aim of this study was a comprehensive assessment of the impact of the anticoagulant type in blood collection tubes on circulating plasma miRNA profiles using small RNA sequencing. Blood from ten healthy participants (five males and five females from 25 to 40 years old) was taken in collection tubes with four different anticoagulants: acid citrate dextrose (ACD-B), sodium citrate, citrate-theophylline-adenosine-dipyridamole (CTAD) and dipotassium-ethylenediaminetetraacetic acid (K2 EDTA). Platelet-free plasma samples were obtained by double centrifugation. EDTA plasma samples had elevated levels of hemolysis compared to samples obtained using other anticoagulants. Small RNA was extracted from plasma samples and small RNA sequencing was performed on the Illumina NextSeq 500 system. A total of 30 samples had been successfully sequenced starting from ~1 M reads mapped to miRNAs, allowing us to analyze their diversity and isoform content. The principal component analysis showed that the EDTA samples have distinct circulating plasma miRNA profiles compared to samples obtained using other anticoagulants. We selected 50 miRNA species that were differentially expressed between the sample groups based on the type of anticoagulant. We found that the EDTA samples had elevated levels of miRNAs which are abundant in red blood cells (RBC) and associated with hemolysis, while the levels of some platelet-specific miRNAs in these samples were lowered. The ratio between RBC-derived and platelet-derived miRNAs differed between the EDTA samples and other sample groups, which was validated by quantitative PCR. This study provides full plasma miRNA profiles of 10 healthy adults, compares them with previous studies and shows that the profile of circulating miRNAs in the EDTA plasma samples is altered primarily due to an increased level of hemolysis.

P-239 Embryonic quality assessment from microRNA profiling

Abstract Study question Does the microRNA expression profile in blastocysts change with different embryonic quality parameters? Summary answer MicroRNA expression showed significant changes when aneuploidy was present, in day-6 compared to day-5 blastocysts and with different trophectoderm (TE) morphology grades. What is known already MicroRNA (miRNA) is a gene expression regulatory mechanism used as a diagnostic marker for some conditions. Despite the great magnitude of studies investigating the miRNA role in implantation and pregnancy complications, limited work has been done to evaluate it as a potential marker for preimplantation embryo quality. With the accelerated changes in gene expression at early embryos, the miRNA profile is a potential indicator of embryo quality. This study investigates the blastocyst miRNA profile in relation to three quality parameters: aneuploidy, the day of blastocyst formation and morphology. Study design, size, duration We collected 122 surplus embryos donated for research by 46 consenting couples from Centre for Reproductive and Genetic Health, London. All samples were ICSI derived blastocysts (day5/6) with PGT-A results. For aneuploidy status comparison, samples were categorized according to the number of chromosomes affected (single/multiple) and type of aneuploidy (gain/loss/partial), compared to euploid. The day of blastocyst development has (day5/day6) groups, and finally samples were compared based on TE morphology (excellent/good/fair). Participants/materials, setting, methods Frozen human embryos were individually thawed and transferred to a lysis buffer. The miRNAs were extracted from each sample then sequenced by next generation sequencing (NGS) using miRNA Library Kit (QIAGEN) with NextSeqTM 500 (illumina). The differential expression analysis was conducted using DESeq2 via GeneGlobe (QIAGEN). We set the significance cut-offs to (-2&amp;gt; foldchange &amp;gt;2) and (&amp;lt;0.05 FDR p-value). For gene ontology and pathway annotation, we used miRTargetLink 2.0 and miRPathDB 2.0 platforms. Main results and the role of chance Compared to the euploid blastocysts, all aneuploid categories, except the single chromosome aneuploidy, had significant differences in miRNA expression. Nine miRNAs were altered in the group with multiple aneuploidies, and one was highly upregulated in embryos with partial losses or gains. The majority of differentially expressed (DE) miRNAs in samples with whole chromosomal gain or loss were downregulated. Conversely, four miRNAs showed a significant increase in day-6 blastocysts compared to day-5. Morphology grade comparisons showed no differences between excellent and good TE; however, six miRNAs were significantly changed in fair TE compared to good. The frequently altered miRNAs in these comparisons were ( hsa-let-7c-5p, hsa-miR-206, hsa-miR-184 and hsa-miR-203a-3p ). Unsurprisingly, pathway annotation of the DE miRNAs showed an involvement of key biological processes, like apoptosis, cell-cycle and differentiation, signalling and gene expression regulation, in all groups. The pathways of note in aneuploid blastocysts included DNA damage response, intrinsic and extrinsic apoptotic signalling, and regulation of cell-cycle G1/S associated events. In blastocyst day5/day6 comparison, embryonic and organ development were the most involved pathways, while in TE morphology, kinase activity and phosphorylation along with apoptosis and DNA damage response were significant. Limitations, reasons for caution Differential expression analysis based on inner cell mass (ICM) morphology and blastocyst expansion were not applicable due to the inadequate number of embryos per group; as all the samples collected were in excellent or good quality. Wider implications of the findings Gene expression profiling of miRNAs in human blastocysts will potentially identify biomarkers predictive of implantation success. Trial registration number not applicable

Rolling Circle and Loop Mediated Isothermal Amplification Strategy for Ultrasensitive miRNA Detection

Rolling circle amplification (RCA) and loop mediated isothermal amplification (LAMP) were combined to establish the rolling circle and loop mediated isothermal amplification (RC-LAMP) method for miRNA detection. With the participation of Bst 2.0 DNA Polymerase, the method enabled RCA and LAMP amplification to occur simultaneously without thermal cycling. The limit of detection of RC-LAMP was 500 amol/L, which is comparable to previously reported amplification strategies. Moreover, its upper limit of quantitation was higher and showed a stronger resistance to matrix interference. Therefore, it is possible to detect low concentrations of miRNA in samples by increasing the total RNA added. Owing to its facile detection mode and simple operation, this method has great potential in clinical sample detection.

Pap Smear miR-92a-5p and miR-155-5p as potential diagnostic biomarkers of squamous intraepithelial cervical cancer.

Background:one of the female-specific diseases with a high incidence and mortality is cervical cancer. The main cause of cervical cancer is infection with Human papilloma virus (HPV). Low-grade squamous intraepithelial lesions (LSIL) and High-grade squamous intraepithelial lesions (HSIL) usually is caused by an HPV infection. Considering the role of microRNAs (miRNAs) as diagnostic biomarkers for a variety of cancers, the aim of this study was to determine miR-92a-5p and miR-155-5p expression levels in LSIL and HSIL Pap Smear samples. Methods:After initial bioinformatic studies, A total of 75 samples (25 samples of patients with LSIL, 25 patients with HSIL and 25 healthy individuals) were subjected to RNA extraction and cDNA synthesis. The expressions levels of confirmed miRNAs in samples of patients with LSIL, HSIL and healthy individuals were evaluated by Real time PCR analysis. To demonstration the role of predicted miRNAs as novel biomarkers in diagnosis of LSIL and HSIL, ROC curve analysis was done. Results:Bioinformatics results showed that miR-92a-5p and miR-155-5p target the HPV E6 and E7 genes. The expression levels of these miRNAs were strikingly higher in Pap smear of patients with LSIL than in the healthy individuals (35.36, P = 0.001) (62.23, P = 0.001). Similarity, expression levels of miR-92a-5p and miR-155-5p were amazingly higher in patients with HSIL than in the healthy individuals (33.62, P= 0.001) (69.07, P= 0.001). Although, the levels of miR-92a-5p (0.95, P = 0. 85) and miR-155-5p (1.11, P = 0.84) exhibited no statistical differences between patients with LSIL and HSIL. Also, ROC curve analyses verified that miR-92a-5p and miR-155-5p are specific and sensitive and may serve as new biomarkers for the early detection of cervical cancer. Conclusion:These data suggest miR-92a-5p and miR-155-5p, which are upregulated in LSIL and HSIL, can be consider as predictive biomarkers for the prognosis of cervical cancer patients.

Distance-based quantification of miRNA-21 by the coffee-ring effect using paper devices.

Enabled by the coffee-ring effect, a paper-based signal transduce method is employed for catalytic hairpin assembly (CHA) amplification and hybridization chain reaction (HCR) to achieve miRNA quantification. Once the target miRNAs appeared, it was circularly used by CHA to initiate HCR amplification to produce a large number of G-quadruplex, which is combined with hemin to form a hemin/G-quadruplex DNAzyme. The DNAzyme catalyzes a colorimetric reaction to produce colored nanoparticles, which were converted to the end edge of the paper by evaporation-driven flow, forming a visible colored band. Higher concentration of miRNA led to more colored nanoparticles and thus a longer colored band that can simply be measured by a ruler. The results of determination of miRNA in samples demonstrate that the relative standard deviation of the proposed approach is 5.2%, highly sensitive and repeatable, with a working range 1.0 to 1000 pM and a LOD of 0.2 pM. The paper-based analytical device as a novel platform offers a new signal transduce pathway toward the detection of low-abundance biomarkers for diagnosis.Graphical abstract Schematic representation of the principle for quantification of miRNA on paper based on the coffee-ring effect.

A novel transthyretin/STAT4/miR-223-3p/FBXW7 signaling pathway affects neovascularization in diabetic retinopathy

MicroRNAs (miRNAs) are small RNAs without protein-coding functions that negatively regulate target genes and play important roles in physiological and pathological processes. The aim of this work was to reveal a novel miRNA/gene pathway in diabetic retinopathy (DR). A microarray was used to screen miRNAs in samples from nondiabetic controls and patients with DR, and miR-223-3p was screened as a potential candidate. Quantitative real-time PCR (qRT-PCR) revealed that the level of miR-223-3p was frequently overexpressed in DR samples and human retinal endothelial cells (hRECs) in hyperglycemia, but it was decreased in hyperglycemia after the addition of transthyretin (TTR). In addition, according to cell proliferation, tube formation, and wound healing assays, the downregulation of miR-223-3p suppressed cell migration and proliferation, whereas miR-223-3p upregulation showed the opposite effects. Furthermore, luciferase assays identified F-box and WD repeat domain-containing 7 (FBXW7) as a target mRNA of miR-223-3p. High glucose conditions facilitated the recruitment of signal transducer and activator of transcription 4 (STAT4) and promoted the transcription of miR-223-3p. In hRECs, in a hyperglycemic environment, TTR inhibited STAT4 expression, downregulated the level of miR-223-3p, and finally promoted FBXW7 expression. This study found a novel mechanism whereby TTR might affect neovascularization through a newly identified STAT4/miR-223-3p/FBXW7 cascade in DR.

MiRNA expression patterns are associated with tumor mutational burden in lung adenocarcinoma

ABSTRACTBackground: Tumor mutational burden (TMB) has emerged as an independent biomarker to predict patient responses to treatment with immune checkpoint inhibitors (ICIs) for lung adenocarcinoma (LUAD). MicroRNAs (miRNAs) have a crucial role in the regulation of anticancer immune responses, but the association of miRNA expression patterns and TMB is not clear in LUAD.Methods: Differentially expressed miRNAs in samples with high TMB and low TMB samples were screened in the LUAD dataset in The Cancer Genome Atlas. The least absolute shrinkage and selection operator (LASSO) method was applied to develop a miRNA-based signature classifier for predicting TMB levels in the training set. An test set was used to validate this classifier. The correlation between the miRNA-based classifier index and the expression of three immune checkpoints (PD-1, PD-L1, and CTLA-4) were explored. Functional enrichment analysis was carried out of the miRNAs included in the miRNA-based signature classifier.Results: Twenty-five differentially expressed miRNAs were used to establish a miRNA-based signature classifier for predicting TMB level. The accuracy of the 25-miRNA-based signature classifier was 0.850 in the training set, 0.810 in the test set and 0.840 in the total set. This miRNA-based signature classifier index showed a low correlation with PD-1 and PD-L1, and no correlation with CTLA-4. Enrichment analysis for these 25 miRNA revealed they are involved in many immune-related biological processes and cancer-related pathways.Conclusion: MiRNA expression patterns are associated with tumor mutational burden and a miRNA-based signature classifier may serve as a biomarker for prediction of TMB levels in LUAD.

Host miRNA Profile of Aspergillus Colonization Resulting in CLAD in Lung Transplant Recipients

Purpose Chronic lung allograft dysfunction (CLAD) has been associated with Aspergillus colonization after lung transplantation . We sought to identify the miRNA profile that distinguishes Aspergillus colonization with CLAD from Aspergillus colonization without CLAD in bronchoalveolar lavages (BALs) collected from lung transplant recipients (LTRs). Methods We used the Nanostring nCounter Human miRNA v3 panel to measure human miRNAs in BALs collected from LTRs with Aspergillus colonization and CLAD (n=7) against those with Aspergillus colonization without CLAD (n=10) and control stable LTRs without Aspergillus colonization (n=9). The miRNA profile was compared using the permutation test of 100000 trials for each of the comparisons. We used mirDip ( http://ophid.utoronto.ca/mirDIP/index.jsp#r ) to obtain their gene targets. The targets were filtered by their integrated score to be greater or equal to 90%. Subsequently, we used pathDIP ( http://ophid.utoronto.ca/pathDIP/ ) to determine pathway enrichment. Results We found 92 differentially expressed miRNAs in samples from Aspergillus colonization with CLAD compared to colonization without CLAD and controls (p-value = 90% resulted in 8 miRNAs which targeted 320 genes. These miRNAs were differentially expressed by more than two folds, and included hsa-miR-195-5p, hsa-miR-214-3p, hsa-miR-199b-5p, hsa-miR-424-5p, hsa-miR-181c-5p, hsa-miR-145-5p, hsa-miR-32-3p and hsa-miR-128-3p (Figure 1b). Conclusion This pilot study suggests that patients with Aspergillus colonization with CLAD may have a distinctive miRNA profile compared to patients with Aspergillus colonization without CLAD. Further longitudinal studies are required to evaluate whether these miRNAs can help identify Aspergillus colonized patients who may later develop CLAD.

MP28-11 A 4-MIRNA SCORE PREDICTS THE INDIVIDUAL METASTATIC RISK OF RENAL CELL CARCINOMA PATIENTS

You have accessJournal of UrologyKidney Cancer: Epidemiology & Evaluation/Staging I1 Apr 2018MP28-11 A 4-MIRNA SCORE PREDICTS THE INDIVIDUAL METASTATIC RISK OF RENAL CELL CARCINOMA PATIENTS Joana Heinzelmann, Madeleine Arndt, Ramona Pleyers, Tobias Fehlmann, Sebstian Hölters, MArtin Janssen, Alexey Pryalukhin, Rainer Bohle, Mieczyslaw Gajda, Elke Schaeffeler, Michael Stöckle, and Kerstin Junker Joana HeinzelmannJoana Heinzelmann More articles by this author , Madeleine ArndtMadeleine Arndt More articles by this author , Ramona PleyersRamona Pleyers More articles by this author , Tobias FehlmannTobias Fehlmann More articles by this author , Sebstian HöltersSebstian Hölters More articles by this author , MArtin JanssenMArtin Janssen More articles by this author , Alexey PryalukhinAlexey Pryalukhin More articles by this author , Rainer BohleRainer Bohle More articles by this author , Mieczyslaw GajdaMieczyslaw Gajda More articles by this author , Elke SchaeffelerElke Schaeffeler More articles by this author , Michael StöckleMichael Stöckle More articles by this author , and Kerstin JunkerKerstin Junker More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.911AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The prognosis of patients with renal cell carcinoma strongly depends on the metastatic potential of the primary tumor. Recently, we have identified miRNAs which characterize primary renal cell carcinoma (ccRCC) with high metastatic risk. The aim of this study is to determine whether a combination of differently expressed miRNAs in samples of primary resected tumors can predict the individual risk for metastatic disease in ccRCC patients. METHODS FPE samples of metastatic (n=57) and non-metastatic (n=51) primary ccRCCs were collected between 2000 and 2013 in two German academic centers. We evaluated the association of altered miRNA expression with the metastatic potential of tumors using qPCR. In a test-cohort, we selected the best discriminators for metastatic and non-metastatic ccRCC. The selected miRNAs were subsequently approved in a validation- cohort. A prognostic 4-miRNA-signature was established using support vector machines. Cox regression analyses were performed using IBM SPSS Statistics for correlation of miRNA-model and clinico-pathological parameters to metastasis-free and overall survival. RESULTS Nine out of 14 candidate miRNAs showed significant expression alterations in the test cohort. Thereof, eight miRNAs were approved in the validation cohort. We established a 4-miRNA score (miR-30a-3p, miR-30c-5p, miR-139-5p, miR-144) with a sensitivity of 86%, a specificity of 70% and a accuracy of 78%. This score was the superior predictor for metastasis-free (HR=13.614, p=0.00003) and overall survival (p=0.002) compared to T-category, Grading and N-status, likewise in the subgroup of patients with low risk tumors (pT1a/b, cN0, cM0, G1-2) (HR=17.323, p=0.028). CONCLUSIONS The 4-miRNA score is applicable as an independent prognostic molecular tool superior to clinico-pathological parameters for accurate prediction of individual metastatic risk even in low risk ccRCC. Our score improves the stratification of ccRCC patients in high and low metastatic risk groups for individual follow-up and adjuvant therapy strategies. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e360 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Joana Heinzelmann More articles by this author Madeleine Arndt More articles by this author Ramona Pleyers More articles by this author Tobias Fehlmann More articles by this author Sebstian Hölters More articles by this author MArtin Janssen More articles by this author Alexey Pryalukhin More articles by this author Rainer Bohle More articles by this author Mieczyslaw Gajda More articles by this author Elke Schaeffeler More articles by this author Michael Stöckle More articles by this author Kerstin Junker More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Independent prognostic miRNAs for bladder urothelial carcinoma.

Bladder cancer is the most common malignant tumor of the urinary system, and it is also an important cause of death by cancer globally. Increasing number of studies have shown that miRNAs can be used as prognostic markers for cancers. This study made use of the data available in the Cancer Genome Atlas in order to statistically analyze reported expression levels of miRNAs in samples from bladder urothelial carcinoma patients. Clinical features from a total of 399 patients and the expression data for 1,581 kinds of miRNA were included in the study. Single factor Cox regression analysis was used to identify miRNAs related to survival times for the patients. Then, through multifactors Cox regression we sorted out the independent prognostic miRNAs for the carcinoma. According to our results, 19 miRNAs were closely related to the survival times of patients with bladder urothelial carcinoma, and 3 miRNAs including hsa-mir-518b (p=0.02), hsa-mir-192 (p=0.04) and hsa-mir-7705 (p=0.04) should be useful as independent prognostic factors in patients. In addition, the survival time of those expressing high levels of hsa-mir-7705 and hsa-mir-192 was less than the survival time of those with low expression levels. However, there were no obvious differences in the survival times between high and low expressors of hsa-mir-518b. According to our results, hsa-mir-7705, hsa-mir-192 and hsa-mir-518b can be applied as independent prognostic markers for bladder urothelial carcinoma.

554P Study on the differentially expressed miRNA of drug- contained serum of Bushen decoction on breast cancer bearing nude mice osteoblast by miRNA array

Background This study aimed to investigate the effect on the target or related genes of miRNA after serum containing Bushen decoction intervention on breast cancer-bearing nude mice osteoblast cells, at the differential expression of miRNA profile levels. Methods The primary breast cancer-bearing nude mice osteoblasts were identified by alkaline phosphatase staining and collagen type I immunofluorescent staining and mineralized nodules staining. The osteoblasts were cultured in serum containing Bushen decoction. After a given time of intervention, the cultured cells were collected, and the total RNA were isolated and purified and used for follow-up miRNA microarray experiments. Real-time quantitative PCR was used to further validate the expression level of miRNA in samples of Bushen decoction intervention on breast cancer-bearing nude mice osteoblasts. Results The twice-enzyme digestion method was practicable, efficient and ideal technique to obtain a large number of purified primary breast cancer-bearing nude mice osteoblasts in a short time and culture cells with typical osteoblast characteristics. Alkaline phosphatase staining and collagen type I immunofluorescent staining and mineralized nodules staining were positive. Compared with the Bushen decoction containing serum cultured 1d, 229 different expression of miRNA (52 up-regulated and 177 down-regulated more than twice) were found 6 days after Bushen decoction containing serum cultured. The results of real-time PCR were in accordance with the miRNA array results that the expression levels of mmu-miR-205 was over 4-fold up- regulated and mmu-miR-335 over 4-fold down-regulated significantly in the cultured 6d compared with the cultured 1d. Conclusions A set of specific miRNA were obtained after Bushen decoction containing serum intervention on breast cancer-bearing nude mice osteoblast cells for 6d. Target gene prediction software predicts the up-regulated miRNA mainly by regulating bone development, bone morphogenesis, bone resorption and endochondral bone morphogenesis through the target genes, such as Runx2, Wnt, Notch, and Axin2, et al. Legal entity responsible for the study N/A Funding N/A Disclosure All authors have declared no conflicts of interest.

Lessons from microRNA Sequencing Using Illumina Technology

The numbers of reads generated by second-generation sequencing technologies permit to establish in a single sequencing lane multiple microRNA (miRNA) expression profiles from small RNA-derived cDNA libraries tagged by barcodes consisting of few bases. Multiplex sequencing allows sample size expansion and thus the statistical reliability of generated data. This allows the detection of discrete changes in miRNA expression levels that occur at the onset of cellular processes. With the development of the “by-amplification” strategy, tagging cDNA libraries is no more a source of technical variability. However, other specific features should be kept in mind when designing experiments aimed at profiling miRNA expression using Illumina sequencing technology, the most important being the substantial distortion between miRNA expression in sequencing data and the true miRNA abundancy. miRNAs of low expression in profiles may correspond to abundant miRNAs in samples and vice versa. We report here data obtained from rat cerebellum and liver that illustrate 1) the high 3’ adaptor dependency of miRNA expression profiles, 2) the impact of sample size when working with moderate (3 - 4 fold) changes of miRNA expression and 3) the impact of the statistical tools used to identify differentially expressed miRNAs.

P67: Use of extracellular RNA from urine for the diagnosis of prostate cancer

Differently sized microparticles, including exosomes (30–100 nm), prostasomes (50–500 nm), oncosomes (50–500 nm) and other microparticles (100–1000 nm) were found in blood and urine. Exosomes from prostate cancer (PCa) patients can potentially contain cancer-specific nucleic acids, and thus can represent a valuable source of diagnostic material. In this study, we have investigated microvesicles and miRNA from urine of healthy donors and PCa patients. To isolate miRNAs from urine and microparticles, novel methods for miRNA isolation were elaborated (Rus. patent application No 2014137763, priority date 17.09.2014). The study population included 14 patients with PCa (63–82 years, T2-3NxMx1) and control group of 20 healthy volunteers with no previous history of prostate disease (48–73 years). Urine was clarified by two serial centrifugations at 400 g , 20 °C, 20 min and at 17000 g , 20 °C, 20 min. Microparticles were precipitated from the resulting supernatant by high-speed centrifugation at 100000 g , 18 °C, 90 min, the pellet was resuspended and pelleted by centrifugation under the same conditions. To isolate exosomes, total microparticles were filtered through 0.1 μm pore filters and reprecipitated. The resulting pellets were resuspended and exosome samples were investigated by transmission electron microscopy (TEM). MiRNAs were isolated by one-step single-phase protocol and purified using “BioSilica” spin- columns (Zaporozhchenko et al., Anal. Biochem, upcoming, doi: 10.1016/j.ab.2015.03.028) and by recently developed method based on precipitation of excess biopolymers, allowing to isolate miRNAs with better efficiency than commercially available kits. The size and quantity assessment of extracellular RNA were performed using capillary electrophoresis system on Agilent 2100 Bioanalyzer. Concentrations of miRNAs (miR19b, miR25, miR205, miR125b, miR126) were measured by qRT-PCR and normalized to miR-16 using dCq method. TEM demonstrated the presence of 20–300 nm microparticles in urine of healthy donors and PCa patients. Approximately 50–70% of all urine microparticles are represented by 30–100 nm exosomes and residual 30–50% by particles larger than 100 nm. (The pool of urine microvesicles consists of 50–70% exosomes and 30–50% particles larger than 100 nm.). The major part of extracellular RNA found both in exosomes and total microparticles fraction of healthy donors and patients with PCa, is 25–200 nt long and can include tRNA (73–93 n.), 5.8 rRNA (∼150 n.), snoRNK (10–20 n.), snRNA (60–300 n.) piRNAs (29–30 n.), miRNAs (20–25 n.), siRNA (21–25 n.). Concentration of extracellular urine RNA in exosomes and total microparticles of healthy donors and patients with PCa amounts to100 pg/ml of urine on average.Receiver Operating Characteristic (ROC) curve analysis of all miRNAs in samples isolated from total urine did not demonstrate any diagnostic significance. In contrast, in training cohort concentration of miR-19b in exosomes and total microparticles fraction provide 95%/75% and 93%/100% sensitivity and specificity, respectively. Thus, microparticles, isolated from urine of PCa patients represent a valuable source of diagnostically significant miRNAs. To investigate miR-19b, a large testing cohort is required. Search for other potential miRNA-markers by microarray profiling of cell-free miRNA isolated from urine of PCa patients is also required to reveal a sold set of markers for confident PCa diagnostics.

Abstract 3387: A complete workflow for high throughput isolation of serum microRNAs and downstream analysis by qRT-PCR: application to cancer biomarker discovery

Abstract Biomarkers have become invaluable tools in the clinical space for cancer detection, diagnosis, patient prognosis and treatment selection. An ideal biomarker should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. Although many candidate biomarkers for various diseases have been proposed in the literature, very few have made their way to clinical use, and for certain types of cancer there are no reliable biomarker options. The use of prostate specific antigen (PSA) has led to a dramatic increase in the diagnosis of prostate cancer - over 230,000 new cases in 2014. However, most of these cases are of low risk disease that is unlikely to cause significant morbidity and mortality, leading to the accepted notion that prostate cancer is overdiagnosed. On the other hand, while it is a sensitive test, less than ¼ of patients undergoing a prostate biopsy based on PSA testing are actually found to have the disease. The identification of biomarkers that are more senstitive and specific predictors for the presence of prostate cancer, as well as for the aggressiveness of the disease, could decrease the complications and morbidity related to prostate biopsies and definitive treatment. The circulating miRNA in blood could potentially fill this void, providing biomarkers suitable for real-time detection and application to the management of prostate cancer patients. The main advantages of miRNAs versus longer molecules (mRNA, lncRNA) include: (i) smaller number of sequences &amp;lt;2000 and thus ease of analysis (ii) stability due to small size ∼16-27 nt and thus robustness of detection. The objective of this project was to develop a set of reagents and tools for efficient recovery and analysis of circulating miRNAs. Our team has built a complete workflow to provide: (1) high throughput isolation of total serum RNA using a new MagMaxTM mirVanaTM reagent kit for the KingFisherTM Flex and Duo instruments, and (2) downstream analysis of microRNA profiles by qRT-PCR using next generation TaqMan® microRNA assays. The effectiveness of this workflow is exemplified by analysis of a panel of miRNAs in samples derived from serum of patients with metastatic prostate cancer versus control groups. Following this fast and easy workflow suitable for serum and other body fluids, disease-specific RNA signatures can be identified and used as biomarkers. TaqMan® is a registered Trademark of Roche Molecular Systems, Inc. used under permission and license. Citation Format: Emily M. Zeringer, Alex J. Rai, Joel DeCastro, Luming Qu, Marie Gonzalez, Laura Chapman, Alexander V. Vlassov, Susan M. Magdaleno, Chunmei Liu, Fangqi Hu, Shoulian Dong, Linda Wong. A complete workflow for high throughput isolation of serum microRNAs and downstream analysis by qRT-PCR: application to cancer biomarker discovery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2015-3387

Abstract 3988: MicroRNA expression associated with response grade in gastric cancer patients’ tumors

Abstract Background: Many chemotherapy schemes have been used in neoadjuvant treatment scenario to improve the overall and disease-free survival of gastric cancer (GC) patients treated with surgery alone. However, in some cases, the objective clinical response is not achieved, and this lack of response to neoadjuvant chemotherapy may delay curative surgery and increase surgical complications. Therefore, a major challenge for improving clinical outcomes is to understand the molecular mechanisms that may play essential roles in patients responses of GC treatments. The expression levels of several miRNAs have been found to be correlated with GC prognosis and could determine the clinical response for neoadjuvant chemotherapy. Therefore, our goal was to analyze the relation between miRNA profile and the pathological response grade of GC patients tumors treated with neoadjuvant chemotherapy or surgery alone. Methods: We selected 34 biopsy tumor samples of patients treated with neoadjuvant chemotherapy (collected before the treatment) and 23 tumor samples of patients submitted to surgery without adjuvant treatment. All patients treated with neoadjuvant chemotherapy received ECF, DCF or ECX prior the surgery, and subsequently they were submitted to a complete resection. The identification of differentially expressed miRNAs was done by TLDA Quantitative Real Time PCR. Results: After categorizing neoadjuvant chemotherapy patients according to percentage of viable tumor cells present in gastric resection and the surgical patients according to their 5 years of follow up status we could segregate those with and without response for both treatments. The comparison between response grade versus miRNAs showed 9 and 14 downregulated miRNAs in samples of patients without response for neoadjuvant chemotherapy and surgery alone, respectively. We also could observe 3 common downregulated miRNAs between patients without response in both groups. Conclusion: We identified some downregulated miRNAs involved in gastric cancer pathways and mechanisms of drug resistance that should manage to determine a predictable miRNA signature to perform a correct patient selection for GC treatments. Note: This abstract was not presented at the meeting. Citation Format: Michelle Maccarini Barcelos Baldoni, Cláudia Malheiros Coutinho Camillo, Renato David Puga, Maria Dirlei Ferreira Begnami. MicroRNA expression associated with response grade in gastric cancer patients’ tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3988. doi:10.1158/1538-7445.AM2015-3988

MiRNAs As Diagnostic and Prognostic Biomarkers in Pancreatic Ductal Adenocarcinoma and Its Precursor Lesions: A Review.

Pancreatic ductal adenocarcinoma (PDAC), a rare but lethal tumor, is difficult to diagnose without performing an invasive procedure. miRNAs are known to be deregulated in PDAC patients, and recent studies have shown that they can be used as diagnostic and prognostic of the disease. The detection of miRNAs in samples acquired through minimally or noninvasive procedures, such as serum, plasma, and saliva, can have a positive impact on the clinical management of these patients. This article is a comprehensive review of the major studies that have evaluated the expression of miRNAs as biomarkers in pancreatic cancer and its premalignant lesions.