• Polymeric nanoparticles
• apoptosis
• co-delivery
• in vivo animal model
• lung cancer

• A549 Cells
• Animals
• Antineoplastic Agents/administration & dosage
• Antineoplastic Agents/pharmacology
• Apoptosis/drug effects
• Cell Survival/drug effects
• Chemistry, Pharmaceutical
• Crizotinib/administration & dosage
• Crizotinib/pharmacology
• Drug Carriers/chemistry
• Drug Combinations
• Drug Liberation
• Human Umbilical Vein Endothelial Cells
• Humans
• Lung Neoplasms/drug therapy
• Mice, Nude
• Nanoparticles/chemistry
• Polymers/chemistry
• Sorafenib/administration & dosage
• Sorafenib/pharmacology
• Xenograft Model Antitumor Assays
• Mice

• biomarkers
• body fluids
• diagnosis
• imaging
• lung cancer
• predictors

• Biomarkers, Tumor/genetics
• Biomarkers, Tumor/metabolism
• Biopsy
• Diagnostic Imaging
• Disease Progression
• Early Detection of Cancer
• Humans
• Lung Neoplasms/diagnosis
• Lung Neoplasms/etiology
• Lung Neoplasms/genetics
• Lung Neoplasms/metabolism
• Occupational Diseases/diagnosis
• Occupational Diseases/etiology
• Risk Factors
• Smoking/adverse effects
• Smoking/epidemiology

• IT22349 Mitacs

• biomarkers
• breast cancer
• circulating miRNAs
• extraanalytical variability, sensitivity and specificity
• lung cancer
• miRNA signature
• osteosarcoma
• prostate cancer

Previous researchers have demonstrated that microRNA-505 (miR-505) is negatively correlated with progression in various malignancies. However, the detailed function and molecular mechanisms of miR-505 have yet to be completely elucidated in prostate cancer (PCa). The present study initially identified the potential role of miR-505 in PCa using in vitro experiments, and demonstrated that restoration of miR-505 inhibited proliferation, invasion and migration, yet induced cell cycle arrest and promoted apoptosis in PCa cells. The present study also demonstrated that the expression of neuron-glial-related cell adhesion molecule (NRCAM) was markedly upregulated in PCa cells when compared with benign prostate epithelium. A luciferase reporter assay demonstrated that miR-505 directly targeted NRCAM in PCa cells. In addition, NRCAM stimulation antagonized the inhibitory effects of miR-505 on the proliferation, migration, and invasion of PCa cells. Furthermore, lower levels of miR-505 and higher levels of NRCAM may serve as a predictor of worse biochemical recurrence-free survival or disease-free survival in patients with PCa. In conclusion, the present study revealed the inhibitory effects of miR-505 on PCa tumorigenesis, which potentially occur by targeting NRCAM. The combined analysis of NRCAM and miR-505 may predict disease progression in patients with PCa following radical prostatectomy.

The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed that decreased expression of miR-30a-3p and increased expression of miR-210-3p were significantly associated with the presence of distant metastases. miR-210-3p tumor cell specificity was evaluated by in situ hybridization and its biomarker potential was confirmed by ROC curve analysis (AUC = 0.839). Lastly, agreement between miRNA-seq and RT-qPCR for FFPE-derived RNA was evaluated and a high level of concordance was determined. In conclusion, this study has identified and validated metastasis-related miRNAs in LAC.

• FFPE
• lung adenocarcinoma
• metastasis
• miRNA-seq
• microRNA

• Expression profiling
• Massive parallel sequencing
• MicroRNA
• Microarray
• Multiplexing
• Next-generation sequencing
• RT-PCR
• miRNA

• Computational Biology/methods
• Gene Expression Profiling/methods
• Gene Expression Regulation, Neoplastic
• Gene Library
• High-Throughput Nucleotide Sequencing/methods
• Humans
• MicroRNAs/genetics
• Sequence Analysis, RNA/methods

The study aim was to evaluate the association of the expression of serum microribonucleic acid-590 (miR-590) with the risk of lung squamous cell carcinoma (LUSC), clinicopathological staging and prognosis. A total of 237 patients with LUSC and 100 healthy volunteers (control group) were included in the study. Total RNA was extracted from the peripheral blood serum of the subjects, and the expression level of miR-590 was detected by reverse transcription real-time quantitative polymerase chain reaction. The baseline clinicopathological information of LUSC patients was evaluated, and the patients were followed up with the median follow-up of 47 months. Compared with that in the control group, the expression level of serum miR-590 in LUSC patients was significantly decreased [0.532 (0.367- 0.821) vs. 1.63 (0.893–1.347), P<0.001]. The receiver operating characteristic (ROC) curve showed that the value of predicting LUSC risk using miR-590 was high, the area under curve (AUC) was 0.883, and 95% confidence interval (CI) was 0.829–0.934. In addition, the expression level of serum miR-590 was correlated with pathological staging (P=0.022), lymph node metastasis (P=0.012), distant metastasis (P<0.001) and tumor, node and metastasis (TNM) staging (P=0.044). The overall survival (OS) of patients in the serum miR-590 low expression group was significantly lower than that of the serum miR-590 high expression group (P=0.012), and the low expression of miR-590 was an independent risk factor for the prognosis of patients [hazard ratio (HR)=2.152, 95% CI=1.285–3.233, P=0.004]. The results suggested that the expression level of miR-590 can be used as a biomarker for the risk of disease, disease staging and prognosis of LUSC patients.

• lung cancer
• lung squamous cell carcinoma
• miR-590
• prognosis

At present, cardiovascular diseases are depicted to be the leading cause of death worldwide according to the World Health Organization. In the future, projections predict that ischemic heart disease will persist in the top main causes of illness. Within this alarming context, some tiny master regulators of gene expression programs, namely, microRNAs (miRNAs) carry three promising potentials. In fact, miRNAs can prove to be useful not only in terms of biomarkers allowing heart injury detection but also in terms of therapeutics to overcome limitations of past strategies and treat the lesions. In a more creative approach, they can even be used in the area of human engineered cardiac tissues as maturation tools for cardiomyocytes (CMs) derived from pluripotent stem cell. Very promising not only for patient-specific cell-based therapies but also to develop biomimetic microsystems for disease modeling and drug screening, these cells greatly contribute to personalized medicine. To get into the heart of the matter, the focus of this review lies primarily on miRNAs as acute myocardial infarction (AMI) biomarkers. Only large cohort studies comprising over 100 individuals to reach a potent statistical value were considered. Certain miRNAs appeared to possibly complement protein-based biomarkers and classical risk factors. Some were even described to bear potential in the discrimination of similar symptomatic pathologies. However, differences between pre-analytical and analytical approaches substantially influenced miRNA data. Further supported by meta-analysis studies, this problem had to be addressed. A detailed critical analysis of each step to define miRNAs biomarker potential is provided to inspire a future improved universal strategy. Interestingly, a recurrent set of cardiomyocyte-enriched miRNAs was found, namely, miR-1; miR-133; miR-208a/b; and miR-499a. Each member of this myomiRs group displayed promising roles either individually or in combination as AMI diagnostic or prognostic biomarkers. Furthermore, a precise combo was shown to be powerful enough to transdifferentiate human fibroblasts into CMs opening doors in the therapeutics. Following these discoveries, they also emerged as optional tools to transfect in order to mature CMs derived from pluripotent stem cells. Ultimately, the multiple potentials carried by the myomiRs miR-1; miR-133; miR-208a/b; and miR-499a still remain to be fully unveiled.

• acute myocardial infarction
• cardiac biomarkers
• cardiomyocytes maturation
• microRNAs
• regenerative medicine

At least seven studies have suggested that microRNA levels in whole blood can be diagnostic for lung cancer. We conducted a large bi-institutional study to validate this. Qiagen^® PAXgene^™ Blood miRNA System was used to collect blood and extract RNA from it for 85 pathologic stage I-IV non-small cell lung cancer (NSCLC) cases and 76 clinically-relevant controls who had a benign pulmonary mass, or a high risk of developing lung cancer because of a history of cigarette smoking or age >60 years. Cases and controls were similar for age, gender, race, and blood hemoglobin and leukocyte but not platelet levels (0.23 and 0.26 million/μl, respectively; t test P = 0.01). Exiqon^® MiRCURY^™ microarrays were used to quantify microRNAs in RNA isolates. Quantification was also performed using Taqman^™ microRNA reverse transcription (RT)-PCR assays for five microRNAs whose lung cancer-diagnostic potential had been suggested in seven published studies. Of the 1,941 human mature microRNAs detectable with the microarray platform, 598 (31%) were identified as expressed and reliably quantified among the study's subjects. However, none of the microRNAs was differentially expressed between cases and controls (P >0.05 at false discovery rate <5% in test using empirical Bayes-moderated t statistics). In classification analyses with leave-one-out internal cross-validation, cases and controls could be identified by microRNA expression with 47% and 50% accuracy with support vector machines and top-scoring pair methods, respectively. Cases and controls did not differ for RT-PCR-based measurements of any of the five microRNAs whose biomarker potential had been suggested by seven previous studies. Additionally, no difference for microRNA expression was noticed in microarray-based microRNA profiles of whole blood of 12 stage IA-IIIB NSCLC cases before and three-four weeks after tumor resection. These findings show that whole blood microRNA expression profiles lack diagnostic value for high-risk screening of NSCLC, though such value may exist for selective sub-groups of NSCLC and control populations.

• Adult
• Aged
• Aged, 80 and over
• Biomarkers, Tumor/blood
• Carcinoma, Non-Small-Cell Lung/blood
• Carcinoma, Non-Small-Cell Lung/diagnosis
• Case-Control Studies
• Female
• Humans
• Lung Neoplasms/blood
• Lung Neoplasms/diagnosis
• Male
• MicroRNAs/blood
• Middle Aged
• Oligonucleotide Array Sequence Analysis
• Reproducibility of Results
• Reverse Transcriptase Polymerase Chain Reaction
• Sensitivity and Specificity

• K07 CA111952 NCI NIH HHS
• P30 CA016056 NCI NIH HHS
• R21 CA156087 NCI NIH HHS
• CHEST Foundation
• National Cancer Institute

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases. Absence of miR-103 has recently been identified to be associated with metastatic capacity of primary lung tumors. However, the exact role of miR-103 in NSCLC and the molecular mechanism are unclear. In the present study, we showed that miR-103 expression was reduced in NSCLC tissues and cells. miR-103 expression was negatively correlated with tumor size and stage. The overall survival was longer in patients with higher miR-103 level than in those with lower miR-103 expression. miR-103 inhibited cell proliferation in A549 cells, decreased tumor weight and volume, and prolonged survival of tumor-implanted nude mice. miR-103 increased apoptotic cell death in A549 cells. Furthermore, miR-103 decreased the invasion and migration abilities in A549 cells, as evidenced by Transwell and wound healing results. Downregulation of miR-103 significantly reduced the level of programmed cell death 10 (PDCD10). We found a significant decrease in the relative luciferase activity of the reporter gene in A549 cells cotransfected with the miR-103 mimic and pGL3-PDCD10 WT 3′-UTR, but not pGL3-PDCD10 mut 3′-UTR. We showed that overexpression of PDCD10 significantly inhibited miR-103-induced inhibition of cell proliferation, increased apoptosis, and decreased invasion and migration in A549 cells. Moreover, we found that PDCD10 expression was increased in NSCLC tissues and cells. PDCD10 expression was positively correlated with tumor size and stage. Overexpression of PDCD10 increased cell proliferation and inhibited apoptosis in A549 cells. The data demonstrated that dysregulation of the miR-103/PDCD10 signal may be a novel therapeutic target for the treatment of NSCLC.

Although microRNAs have been recognized as central cellular regulators, there is an evident lack of knowledge about their targets. Here, we analyzed potential target genes for miR-148a functioning in Ras signaling in B cells, including SOS1 and SOS2. A dual-luciferase reporter assay showed significantly decreased luciferase activity upon ectopic overexpression of miR-148a in HEK-293T cells that were co-transfected with the 3′UTR of either SOS1 or SOS2. Each of the 3′UTRs of SOS1 and SOS2 contained two binding sites for miR-148a both of which were necessary for the decreased luciferase activity. MiR-148a overexpression in HEK-293T lead to significantly reduced levels of both endogenous SOS1 and SOS2 proteins. Likewise, reduced levels of SOS proteins were found in two B cell lines that were transfected with miR-148a. The level of ERK1/2 phosphorylation as one of the most relevant downstream members of the Ras/ERK signaling pathway was also reduced in cells with miR-148a overexpression. The data show that miR-148a impairs the Ras/ERK signaling pathway via SOS1 and SOS2 proteins in B cells.

• B cells
• ERK
• SOS
• miR-148a
• microRNA

We present the first sequencing data using the combinatorial probe-anchor synthesis (cPAS)-based BGISEQ-500 sequencer. Applying cPAS, we investigated the repertoire of human small non-coding RNAs and compared it to other techniques.

Starting with repeated measurements of different specimens including solid tissues (brain and heart) and blood, we generated a median of 30.1 million reads per sample. 24.1 million mapped to the human genome and 23.3 million to the miRBase. Among six technical replicates of brain samples, we observed a median correlation of 0.98. Comparing BGISEQ-500 to HiSeq, we calculated a correlation of 0.75. The comparability to microarrays was similar for both BGISEQ-500 and HiSeq with the first one showing a correlation of 0.58 and the latter one correlation of 0.6. As for a potential bias in the detected expression distribution in blood cells, 98.6% of HiSeq reads versus 93.1% of BGISEQ-500 reads match to the 10 miRNAs with highest read count. After using miRDeep2 and employing stringent selection criteria for predicting new miRNAs, we detected 74 high-likely candidates in the cPAS sequencing reads prevalent in solid tissues and 36 candidates prevalent in blood.

While there is apparently no ideal platform for all challenges of miRNome analyses, cPAS shows high technical reproducibility and supplements the hitherto available platforms.

The online version of this article (doi:10.1186/s13148-016-0287-1) contains supplementary material, which is available to authorized users.

• BGISEQ
• Biomarker discovery
• Next-generation sequencing
• miRNA

• Anopheles funestus
• Malaria
• MicroRNA
• Non-coding RNA
• Vector-borne diseases

• Africa
• Animals
• Anopheles/genetics
• Anopheles/growth & development
• Female
• Gene Expression Profiling
• High-Throughput Nucleotide Sequencing
• Life Cycle Stages
• MicroRNAs/biosynthesis
• MicroRNAs/genetics
• Mosquito Vectors/genetics
• Mosquito Vectors/growth & development

• South African National Research Foundation

Transfer RNAs (tRNAs, key molecules in protein synthesis) have not been investigated as potential prognostic markers in breast cancer (BC), despite early findings of their dysregulation and diagnostic potential. We aim to comprehensively profile tRNAs from breast tissues and to evaluate their role as prognostic markers (Overall Survival, OS and Recurrence Free Survival, RFS). tRNAs were profiled from 11 normal breast and 104 breast tumor tissues using next generation sequencing. We adopted a Case-control (CC) and Case-Only (CO) association study designs. Risk scores constructed from tRNAs were subjected to univariate and multivariate Cox-proportional hazards regression to investigate their prognostic value. Of the 571 tRNAs profiled, 76 were differentially expressed (DE) and three were significant for OS in the CC approach. We identified an additional 11 tRNAs associated with OS and 14 tRNAs as significant for RFS in the CO approach, indicating that CC alone may not capture all discriminatory tRNAs in prognoses. In both the approaches, the risk scores were significant in the multivariate analysis as independent prognostic factors, and patients belonging to high-risk group were associated with poor prognosis. Our results confirmed global up-regulation of tRNAs in BC and identified tRNAs as potential novel prognostic markers for BC.

• Biomarkers
• NSCLC
• TNM stage
• histological classification
• miRNAs

• DDPCR
• Microarray
• miRNA

• Animals
• Gene Expression Profiling/methods
• Gene Library
• High-Throughput Nucleotide Sequencing/methods
• Humans
• MicroRNAs/genetics
• Oligonucleotide Array Sequence Analysis/methods
• Polymerase Chain Reaction/methods

• R01CA161837 NCI NIH HHS

The diagnosis of inflammatory bowel disease (IBD) still remains a clinical challenge and the most accurate diagnostic procedure is a combination of clinical tests including invasive endoscopy. In this study we evaluated whether systematic miRNA expression profiling, in conjunction with machine learning techniques, is suitable as a non-invasive test for the major IBD phenotypes (Crohn's disease (CD) and ulcerative colitis (UC)). Based on microarray technology, expression levels of 863 miRNAs were determined for whole blood samples from 40 CD and 36 UC patients and compared to data from 38 healthy controls (HC). To further discriminate between disease-specific and general inflammation we included miRNA expression data from other inflammatory diseases (inflammation controls (IC): 24 chronic obstructive pulmonary disease (COPD), 23 multiple sclerosis, 38 pancreatitis and 45 sarcoidosis cases) as well as 70 healthy controls from previous studies. Classification problems considering 2, 3 or 4 groups were solved using different types of penalized support vector machines (SVMs). The resulting models were assessed regarding sparsity and performance and a subset was selected for further investigation. Measured by the area under the ROC curve (AUC) the corresponding median holdout-validated accuracy was estimated as ranging from 0.75 to 1.00 (including IC) and 0.89 to 0.98 (excluding IC), respectively. In combination, the corresponding models provide tools for the distinction of CD and UC as well as CD, UC and HC with expected classification error rates of 3.1 and 3.3%, respectively. These results were obtained by incorporating not more than 16 distinct miRNAs. Validated target genes of these miRNAs have been previously described as being related to IBD. For others we observed significant enrichment for IBD susceptibility loci identified in earlier GWAS. These results suggest that the proposed miRNA signature is of relevance for the etiology of IBD. Its diagnostic value, however, should be further evaluated in large, independent, clinically well characterized cohorts.

• biomarker
• diagnosis
• lung cancer
• miRNA
• miRNA profiling
• prognosis

There is evidence of blood-borne miRNA signatures for various human diseases. To dissect the origin of disease-specific miRNA expression in human blood, we separately analyzed the miRNome of different immune cell subtypes, each in lung cancer patients and healthy individuals. Each immune cell type revealed a specific miRNA expression pattern also dependinging on the cell origin, line of defense, and function. The overall expression pattern of each leukocyte subtype showed great similarities between patients and controls. However, for each cell subtype we identified miRNAs that were deregulated in lung cancer patients including hsa-miR-21, a well-known oncomiR associated with poor lung cancer prognosis that was up-regulated in all leukocyte subtype comparisons of cancer versus controls. While the miRNome of cells of the adaptive immune system allowed only a weak separation between patients and controls, cells of the innate immune system allowed perfect or nearly perfect classification. Leukocytes of lung cancer patients show a cancer-specific miRNA expression profile. Our data also show that cancer specific miRNA expression pattern of whole blood samples are not determined by a single cell type. The data indicate that additional blood components, like erythrocytes, platelets, or exosomes might contribute to the disease specificity of a miRNA signature.

• lung squamous cell carcinoma
• microRNA
• sequencing

• Biomarker
• Circulating miRNA
• Diagnosis
• Lung cancer
• miRNA

Lung cancer is the leading cause of cancer mortality worldwide. Its high mortality is due to the poor prognosis of the disease caused by a late disease presentation, tumor heterogeneities within histological subtypes, and the relatively limited understanding of tumor biology. Importantly, lung cancer histological subgroups respond differently to some chemotherapeutic substances and side effects of some therapies appear to vary between subgroups. Biomarkers able to stratify for the subtype of lung cancer, prognosticate the course of disease, or predict the response to treatment are in high demand. In the last decade, microRNAs (miRNAs), measured in resected tumor samples or in fine needle aspirate samples have emerged as biomarkers for tumor diagnosis, prognosis and prediction of response to treatment, due to the ease of their detection and in their extreme specificity. Moreover, miRNAs present in sputum, in plasma, in serum or in whole blood have increasingly been explored in the last five years as less invasive biomarkers for the early detection of cancers. In this review we cover the increasing amounts of data that have accumulated in the last ten years on the use of miRNAs as lung cancer biomarkers.

• Lung cancer
• Non-small cell lung carcinomas
• MicroRNAs
• Biomarkers

In our previous studies we reported a panel of 24 miRNAs that allowed discrimination between blood of lung tumor patients independent of the histological subtype and blood of healthy controls with an accuracy of 95.4% [94.9%-95.9%]. Here, we now separately analyzed the miRNA expression in blood of non-small cell lung cancer (NSCLC), including squamous cell lung cancer and adenocarcinoma, and small cell lung cancer (SCLC) patients.

In total, we examined the expression levels of 1,205 miRNAs in blood samples from 20 patients from each of the three histological groups and determined differentially expressed miRNAs between histological subtypes and metastatic and non-metastatic lung cancer. We further determined the overlap of miRNAs expressed in each subgroup with the 24-miRNA signature of lung tumor patients.

Based on a raw p-value < 0.05, only 18 blood-borne miRNAs were differentially expressed between patients with adenocarcinoma and with squamous cell lung carcinoma, 11 miRNAs between adenocarcinoma and SCLC, and 2 between squamous cell lung carcinoma and SCLC. Likewise, the comparison based on a fold change of 1.5 did not reveal major differences of the blood-borne miRNA expression pattern between NSCLC and SCLC. In addition, we found a large overlap between the blood-borne miRNAs detected in the three histological subgroups and the previously described 24-miRNA signature that separates lung cancer patients form controls. We identified several miRNAs that allowed differentiating between metastatic and non-metastatic tumors both in blood of patients with adenocarcinoma and in blood of patients with SCLC.

There is a common miRNA expression pattern in blood of lung cancer patients that does not allow a reliable further subtyping into NSCLC or SCLC, or into adenocarcinoma and squamous cell lung cancer. The previously described 24-miRNA signature for lung cancer appears not primarily dependent on histological subtypes. However, metastatic adenocarcinoma and SCLC can be predicted with 75% accuracy.

The online version of this article (doi:10.1186/1476-4598-13-202) contains supplementary material, which is available to authorized users.

• blood
• cancer
• circulating biomarkers
• lung cancer
• microRNA
• review

• Biomarkers/blood
• Epidemiologic Methods
• Genomics
• Humans
• Lung Neoplasms/genetics
• Metabolomics
• MicroRNAs/biosynthesis
• MicroRNAs/blood
• MicroRNAs/classification
• Research Design

• R01 DK085714 NIDDK NIH HHS
• U01 CA157703 NCI NIH HHS
• R01DK085714 NIDDK NIH HHS
• CA100606 NCI NIH HHS

• Biomarkers
• Deep sequencing
• Lung cancer
• Therapy
• microRNA

肺癌在生物学上具有侵袭性，并且是癌症相关死亡的主要原因。根据临床特征、预后、对治疗的反应和耐受性，每一例肺癌患者的进展均是独特的。传统上基于毛细管的单基因测序的第一代技术（如Sanger测序法）已被允许大量平行测序且成本更低、通量更高的下一代测序技术（next-generation sequencing, NGS）所替代。与传统方法相比，NGS技术取得显著进步。我们希望这些方法可全面地解释癌症全球图谱，并提供更多信息以满足个体化用药的需求。本综述包括对不同NGS技术的简要说明，NGS在肺癌研究进展中的应用和重要发现的总结，包括对已知靶基因（EGFR 、ALK 和KRAS ）的进一步探索、其它肺癌突变的鉴定和癌症基因组研究的全局协调。

• 肺癌
• 下一代测序
• 全外显子组测序
• 全基因组测序
• 全转录组测序

• Exons/genetics
• Genomics
• High-Throughput Nucleotide Sequencing/methods
• Humans
• Lung Neoplasms/drug therapy
• Lung Neoplasms/genetics
• Molecular Targeted Therapy
• Sequence Analysis, RNA

Apoptosis of lymphocytes governs the response of the immune system to environmental stress and toxic insult. Signaling through the ubiquitously expressed glucocorticoid receptor, stress-induced glucocorticoid hormones induce apoptosis via mechanisms requiring altered gene expression. Several reports have detailed the changes in gene expression mediating glucocorticoid-induced apoptosis of lymphocytes. However, few studies have examined the role of non-coding miRNAs in this essential physiological process. Previously, using hybridization-based gene expression analysis and deep sequencing of small RNAs, we described the prevalent post-transcriptional repression of annotated miRNAs during glucocorticoid-induced apoptosis of lymphocytes. Here, we describe the development of a customized bioinformatics pipeline that facilitates the deep sequencing-mediated discovery of novel glucocorticoid-responsive miRNAs in apoptotic primary lymphocytes. This analysis identifies the potential presence of over 200 novel glucocorticoid-responsive miRNAs. We have validated the expression of two novel glucocorticoid-responsive miRNAs using small RNA-specific qPCR. Furthermore, through the use of Ingenuity Pathways Analysis (IPA) we determined that the putative targets of these novel validated miRNAs are predicted to regulate cell death processes. These findings identify two and predict the presence of additional novel glucocorticoid-responsive miRNAs in the rat transcriptome, suggesting a potential role for both annotated and novel miRNAs in glucocorticoid-induced apoptosis of lymphocytes.

• Biomarkers, Tumor/genetics
• Genome, Human
• High-Throughput Nucleotide Sequencing/methods
• Humans
• Lung Neoplasms/diagnosis
• Lung Neoplasms/genetics
• Lung Neoplasms/therapy
• Precision Medicine

• biomarker
• diagnostics
• early detection
• epigenetics
• genetics
• lung cancer
• screening
• transcriptomics

• Multiple sclerosis
• biomarker
• clinically isolated syndrome
• microRNAs
• microarray
• next-generation sequencing
• real-time polymerase chain reaction

• Biomarkers/blood
• Biomarkers/metabolism
• Computational Biology/methods
• Humans
• MicroRNAs/blood
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Neoplasms/blood
• Neoplasms/diagnosis
• Neoplasms/genetics
• Principal Component Analysis/methods
• Probability
• Transcriptome

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

• Next generation sequencing (NGS)
• RNA-Seq
• library preparation
• quantitative PCR (qPCR)
• second generation sequencing (SGS)

• next-generation sequencing
• genomics
• epigenomics
• transcriptomics
• cardiomyopathy
• heart failure

• bioinformatics
• cancer
• gene expression
• high throughput sequencing
• non-coding RNA

The association of lung cancer with changes in microRNAs in plasma shown in multiple studies suggests a utility for circulating microRNA biomarkers in non-invasive detection of the disease. We examined if presence of lung cancer is reflected in whole blood microRNA expression as well, possibly because of a systemic response. Locked nucleic acid microarrays were used to quantify the global expression of microRNAs in whole blood of 22 patients with lung adenocarcinoma and 23 controls, ten of whom had a radiographically detected non-cancerous lung nodule and the other 13 were at high risk for developing lung cancer because of a smoking history of >20 pack-years. Cases and controls differed significantly for age with a mean difference of 10.7 years, but not for gender, race, smoking history, blood hemoglobin, platelet count, or white blood cell count. Of 1282 quantified human microRNAs, 395 (31%) were identified as expressed in the study’s subjects, with 96 (24%) differentially expressed between cases and controls. Classification analyses of microRNA expression data were performed using linear kernel support vector machines (SVM) and top-scoring pairs (TSP) methods, and classifiers to identify presence of lung adenocarcinoma were internally cross-validated. In leave-one-out cross-validation, the TSP classifiers had sensitivity and specificity of 91% and 100%, respectively. The values with SVM were both 91%. In a Monte Carlo cross-validation, average sensitivity and specificity values were 86% and 97%, respectively, with TSP, and 88% and 89%, respectively, with SVM. MicroRNAs miR-190b, miR-630, miR-942, and miR-1284 were the most frequent constituents of the classifiers generated during the analyses. These results suggest that whole blood microRNA expression profiles can be used to distinguish lung cancer cases from clinically relevant controls. Further studies are needed to validate this observation, including in non-adenocarcinomatous lung cancers, and to clarify upon the confounding effect of age.

Blood-born miRNA signatures have recently been reported for various tumor diseases. Here, we compared the miRNA signature in Wilms tumor patients prior and after preoperative chemotherapy according to SIOP protocol 2001.

We did not find a significant difference between miRNA signature of both groups. However both, Wilms tumor patients prior and after chemotherapy showed a miRNA signature different from healthy controls. The signature of Wilms tumor patients prior to chemotherapy showed an accuracy of 97.5% and of patients after chemotherapy an accuracy of 97.0%, each as compared to healthy controls.

Our results provide evidence for a blood-born Wilms tumor miRNA signature largely independent of four weeks preoperative chemotherapy treatment.

MicroRNAs (miRNAs) are involved in the regulation of multiple cellular processes. Changes of miRNA expression have been linked to the development of various diseases including cancer, but the molecular events leading to these changes at different physiological conditions are not well characterized. Here we examined the intracellular events responsible for the miR-466h-5p activation in mouse cells exposed to glucose deprivation. MiR-466h-5p is a member of the miR-297-669 cluster located in intron 10 of Sfmbt2 gene on mouse chromosome 2 and has a pro-apoptotic role. We showed that the time-dependant activation of miR-466h-5p, miR-669c and the Sfmbt2 gene followed the inhibition of histone deacetylation caused by glucose deprivation-induced oxidative stress. This oxidative stress causes the accumulation of reactive oxygen species (ROS) and depletion of reduced glutathione (GSH) that together inhibited histone deacetylases (HDACs) activity, reduced protein levels of HDAC2 and increased acetylation in miR-466h-5p promoter region, which led to the activation of this miRNA. Based on this study and previous work, we suggest a possible role of miR-466h-5p (and miR 297-669 cluster) in the cells during toxic metabolites accumulation. Improved characterization of the molecular events that lead to the activation of miR-466h-5p may provide a better understanding of the relation between cellular environment and miRNA activation.