Analysis Of microRNA Expression Research Articles

Comparative analysis of MicroRNA expression in dog lungs infected with the H3N2 and H5N1 canine influenza viruses

MicroRNAs, a class of noncoding RNAs 18 to 23 nucleotides (nt) in length, play critical roles in a wide variety of biological processes. The objective of this study was to examine differences in microRNA expression profiles derived from the lungs of beagle dogs infected with the avian-origin H3N2 canine influenza virus (CIV) or the highly pathogenic avian influenza (HPAI) H5N1 virus (canine-origin isolation strain). After dogs were infected with H3N2 or H5N1, microRNA expression in the lungs was assessed using a deep-sequencing approach. To identify the roles of microRNAs in viral pathogenicity and the host immune response, microRNA target genes were predicted, and their functions were analyzed using bioinformatics software. A total of 229 microRNAs were upregulated in the H5N1 infection group compared with those in the H3N2 infection group, and 166 microRNAs were downregulated. MicroRNA target genes in the H5N1 group were more significantly involved in metabolic pathways, such as glycerolipid metabolism and glycerophospholipid metabolism, than those in the H3N2 group. The inhibition of metabolic pathways may lead to appetite loss, weight loss and weakened immunity. Moreover, miR-485, miR-144, miR-133b, miR-4859-5p, miR-6902-3p, miR-7638, miR-1307-3p and miR-1346 were significantly altered microRNAs that potentially led to the inhibition of innate immune pathways and the heightened pathogenicity of H5N1 compared with that of H3N2 in dogs. This study deepens our understanding of the complex relationships among microRNAs, the influenza virus-mediated immune response and immune injury in dogs.

Analysis of microRNA expression in the thymus of Myasthenia Gravis patients opens new research avenues.

In early-onset Myasthenia Gravis (MG) with anti-acetylcholine receptor antibodies, thymic abnormalities associated with ectopic germinal centers are frequent. miRNAs by acting as post-transcriptional regulators are involved in autoimmunity. To investigate the implication of miRNAs in thymic changes associated with early-onset MG, we performed a miRnome study and data were analyzed with different approaches. miRNAs of interest were further investigated by RT-PCR and transfection experiments for functional tests. First, analyzing specific dysregulated miRNAs, we focused our attention on miR-7-5p and miR-125a-5p, and confirmed by RT-PCR their respective down- and up-regulation in MG thymuses. miR-7 was the most down-regulated thymic miRNA in MG and we observed an inverse correlation between its expression and CCL21 mRNA expression. We next showed that miR-7 down-regulation was due to thymic epithelial cells and by transfecting these cells with miR-7, we demonstrated that it controlled CCL21 release. As CCL21 is essential for germinal center development, we suggested that miR-7 could be involved in thymic changes associated with MG. miR-125a was up-regulated in MG thymuses and is of great interest as it is known to regulate FoxP3 expression, and to modulate the different inflammatory signaling pathways. Thanks to this thymic miRnome study, we also showed the specific dysregulation of miRNA clusters. In particular, we observed that miRNAs localized at the extremity of the X chromosome were down-regulated. This effect seemed linked to their close localization to the fragile X mental retardation 1 gene (FMR1) and the DNA methylation status. Altogether, this miRnome analysis demonstrated that specific thymic miRNAs can be associated with MG and provides novel insights into the pathogenesis of MG.

T25. Genome-Wide MicroRNA Expression Analysis of PTSD With Comorbid Depression: A Meta-Analysis of Civilian and Veteran Datasets

T25. Genome-Wide MicroRNA Expression Analysis of PTSD With Comorbid Depression: A Meta-Analysis of Civilian and Veteran Datasets

A multiplatform approach identifies miR-152-3p as a common epigenetically regulated onco-suppressor in prostate cancer targeting TMEM97

BackgroundProstate cancer (PCa) is a major cause of morbidity and mortality in men worldwide. MicroRNAs are globally downregulated in PCa, especially in poorly differentiated tumors. Nonetheless, the underlying mechanisms are still elusive. Herein, using combined analysis of microRNAs expression and genomewide DNA methylation, we aimed to identify epigenetically downregulated microRNAs in PCa.ResultsWe found that miR-152-3p was underexpressed in PCa and that lower expression levels were associated with promoter hypermethylation in accordance with TCGA dataset analysis. Functional in vitro assays suggest that miR-152-3p suppresses cell viability and invasion potential, whereas it promotes cell cycle arrest at S and G2/M phases. Additionally, miR-152-3p expression was associated with longer disease-free survival in PCa patients from TCGA. Finally, TMEM97, which is overexpressed in PCa, was identified as a novel miR-152-3p target gene.ConclusionsOur findings demonstrate the advantages of using a combinatory approach to identify microRNAs downregulated due to aberrant promoter methylation. MiR-152-3p downregulation and promoter methylation was found to be prevalent in primary PCa, which impairs its role in control of cell viability, cell cycle regulation and invasion.

Circulating Micrornas Predict Survival of Patients with Tumors of Glial Origin

The World Health Organization has recently introduced molecular prognostic-diagnostic biomarkers in the classification of Central Nervous System (CNS) tumors. In order to characterize subclasses of tumors that cannot find a precise location in the current classification, and, or cannot be tested because of scant material, it is important to find new molecular biomarkers in tissue and, or biological fluid samples. In this study, we identified serum microRNAs that could serve as biomarkers for the diagnosis and prognosis of patients with tumors of glial origin. We retrospectively analyzed microRNA expression in the serum extracellular vesicles of patients with tumors of glial origin. Extracellular vesicles RNA was analyzed by Nanostring. qRT-PCR confirmed 6 overexpressed microRNAs: hsa-miR-4443, hsa-miR-422a, hsa-miR-494-3p, hsa-miR-502-5p, hsa-miR-520f-3p, and hsa-miR-549a. Hsa-miR-4443 was the only microRNA that showed significant differences in most comparisons. In situ hybridization (ISH), confirmed that our signature was mostly expressed in cancer cells.Importantly, hsa-miR-549a and hsa-miR-502-5p expression predicted prognosis in patients with tumors of glial origin. Although more studies are needed, we demonstrated that serum vesicles microRNA profiles are promising diagnostic and prognostic molecular biomarkers that will find an actual application in the clinical practice of CNS tumors.

Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

BackgroundmicroRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods. These included either: phenol: chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction (column-based; miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II). We then evaluated each extraction method for the quality and quantity of RNA recovered, and the expression of miRNAs and target genes.ResultsWe identified differences between each of the RNA extraction methods in the quantity and quality of RNA samples, and in the analysis of miRNA and target gene expression. For the purposes of consistency in quantity, quality and high recovery of miRNAs from tissues, we identified that Phenol:chloroform phase separation combined with silica column-based solid extraction method was preferable (miRVana microRNA isolation). We also identified a method that is not appropriate for miRNA analysis from tissue samples (Bioline Isolate II). For target gene expression any of the kits could be used to analyse mRNA, but if interested in analysing mRNA and miRNA from the same RNA samples some methods should be avoided.ConclusionsDifferent methods used to isolate miRNAs will yield different results and therefore a robust RNA isolation method is required for reproducibility. Researchers should optimise these methods for their specific application and keep in mind that “total RNA” extraction methods do not isolate all types of RNA equally.

Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum

BackgroundBone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture.MethodsA comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared.ResultsPrimary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF.ConclusionsTraditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

Colorimetric detection of microRNA based on DNAzyme and nuclease-assisted catalytic hairpin assembly signal amplification

Accurate and quantitative analysis of microRNA (miRNA) expression is critical for the diagnostics and theranostics of a disease. Herein, a proof-of-concept of a colorimetric horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) biosensor for miRNA assay based on nuclease-assisted catalytic hairpin assembly (CHA) signal amplification was demonstrated. Duplex-specific nuclease (DSN) was employed to cleave the single-stranded DNA (ssDNA) chimeric probe (CP) on the magnetic bead (MB) surface via hybridization of the CP and target miRNA. The regenerated miRNA can cleave a large number of ssDNA CP to produce CHA initiator sequence fragments. The CP consists of two main regions: a target miRNA recognition DNA sequence at the 5′ end and a CHA initiator (CI) sequence at the 3′ end. The catalyzed assembly process of CHA produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimics the horseradish peroxidase activity, which catalyzes a colorimetric reaction. For the proof-of-concept, microRNA-21 (miR-21) was selected as the model target to authenticate this strategy as a versatile assay platform. The proposed strategy allowed quantitation of the sequence specificity of miRNA-21 with a detection limit of 9.2 fM in a dynamic range from 10 fM–1 nM, with an excellent ability to discriminate the differences in miRNAs. Additionally, the miRNA assay in real samples was satisfactory, thereby confirming its applicability. Therefore, this method exhibited a great potential as a miRNA quantification method in biomedical research and clinical diagnosis.

Genome-Wide Analysis of Gene and microRNA Expression in Diploid and Autotetraploid Paulownia fortunei (Seem) Hemsl. under Drought Stress by Transcriptome, microRNA, and Degradome Sequencing

Drought is a common and recurring climatic condition in many parts of the world, and it can have disastrous impacts on plant growth and development. Many genes involved in the drought response of plants have been identified. Transcriptome, microRNA (miRNA), and degradome analyses are rapid ways of identifying drought-responsive genes. The reference genome sequence of Paulownia fortunei (Seem) Hemsl. is now available, which makes it easier to explore gene expression, transcriptional regulation, and post-transcriptional in this species. In this study, four transcriptome, small RNA, and degradome libraries were sequenced by Illumina sequencing, respectively. A total of 258 genes and 11 miRNAs were identified for drought-responsive genes and miRNAs in P. fortunei. Degradome sequencing detected 28 miRNA target genes that were cleaved by members of nine conserved miRNA families and 12 novel miRNAs. The results here will contribute toward enriching our understanding of the response of Paulownia fortunei trees to drought stress and may provide new direction for further experimental studies related the development of molecular markers, the genetic map construction, and other genomic research projects in Paulownia.

Identification and expression analysis of microRNAs in worker caste termites of Coptotermes formosanus and Reticulitermes speratus

A total of 114 microRNAs (miRNAs) were identified for Coptotermes formosanus workers, while a total of 97 miRNAs were identified for Reticulitermes speratus workers, of which 91 were common in C. formosanus and R. speratus. While the relationship between miRNA expression levels in C. formosanus and R. speratus workers had a strong positive correlation, considerable difference in miRNA expressions between C. formosanus and R. speratus was observed. Among the miRNAs up-regulated in C. formosanus workers, miR-11-3p showed the highest expression increase of 2.30 fold, while miR-13b-3p showed the highest expression increase of 5.16 fold among the up-regulated miRNAs in R. speratus workers. Workers of C. formosanus and R. speratus seem to use different miRNAs, miR-11-3p in C. formosanus or miR-13b-3p in R. speratus, to regulate homeodomain transcription factor genes, araucan, caupolican, and nubbin, basic helix-loop-helix (bHLH) transcription factor genes, spineless and E(spl)m3-HLH, and zinc finger transcription factor gene, castor.

RTP801 is a critical factor in the neurodegeneration process of A53T α-synuclein in a mouse model of Parkinson's disease under chronic restraint stress.

Recently, the incidence of Parkinson's disease has shown a tendency to move to a younger population, linked to the constantly increasing stressors of modern society. However, this relationship remains obscure. Here, we have investigated the contribution of stress and the mechanisms underlying this change. Ten-month-old α-synuclein A53T mice, a model of Parkinson's disease (PD), were treated with chronic restraint stress (CRS) to simulate a PD-sensitive person with constant stress stimulation. PD-like behavioural tests and pathological changes were evaluated. Differentiated PC12-A53T cells were treated with corticosterone in vitro. We used Western blot, microRNA expression analysis, immunofluorescence staining, dual luciferase reporter assay and HPLC electrochemical detection to assess cellular and molecular networks after stress treatment. In vivo, stereotaxic injection of shRNA lentivirus was used to confirm our in vitro results. The protein RTP801 is encoded by DNA-damage-inducible transcript 4, and it was specifically increased in dopaminergic neurons of the substantia nigra after CRS treatment. RTP801 was post-transcriptionally inhibited by the down-regulation of miR-7. Delayed turnover of RTP801, through the inhibition of proteasome degradation also contributed to its high content. Elevated RTP801 blocked autophagy, thus increasing accumulation of oligomeric α-synuclein and aggravating endoplasmic reticulum stress. RTP801 inhibition alleviated the symptoms of neurodegeneration during this process. RTP801 is a promising target for the treatment of PD, especially for PD-sensitive patients who live under increased social pressure. Down-regulation of RTP801 could inhibit the current tendency to an earlier onset of PD.

MicroRNA-219 Reduces Viral Load and Pathologic Changes in Theiler's Virus-Induced Demyelinating Disease.

Analysis of microRNA (miR) expression in the central nervous system white matter of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) revealed a significant reduction of miR-219, a critical regulator of myelin assembly and repair. Restoration of miR-219 expression by intranasal administration of a synthetic miR-219 mimic before disease onset ameliorates clinical disease, reduces neurogliosis, and partially recovers motor and sensorimotor function by negatively regulating proinflammatory cytokines and virus RNA replication. Moreover, RNA sequencing of host lesions showed that miR-219 significantly downregulated two genes essential for the biosynthetic cholesterol pathway, Cyp51 (lanosterol 14-α-demethylase) and Srebf1 (sterol regulatory element-binding protein-1), and reduced cholesterol biosynthesis in infected mice and rat CG-4 glial precursor cells in culture. The change in cholesterol biosynthesis had both anti-inflammatoryand anti-viral effects. Because RNA viruses hijack endoplasmic reticulum double-layered membranes to provide a platform for RNA virus replication and are dependent on endogenous pools of cholesterol, miR-219 interference with cholesterol biosynthesis interfered virus RNA replication. These findings demonstrate that miR-219 inhibits TMEV-induced demyelinating disease through its anti-inflammatory and anti-viral properties.

Association of miR-548c-5p, miR-7-5p, miR-210-3p, miR-128-3p with recurrence in systemically untreated breast cancer.

Current prognostic markers allocate the majority of lymph node (LN) negative and estrogen receptor (ER) positive breast cancer patients into the high-risk group. Accordingly, most patients receive systemic treatments although approximately 40% of these patients may have been cured by surgery and radiotherapy alone. Two studies identified seven prognostic microRNAs in systemically untreated, LN negative and ER positive breast cancer patients which may allow more precise patient classification. However, six of the seven microRNAs were analyzed in both studies but only found to be prognostic in one study. To validate their prognostic potential, we analyzed microRNA expression in an independent cohort (n = 110) using a pair-matched study design minimizing dependence of classical markers. The expression of hsa-miR-548c-5p was significantly associated with abridged disease-free survival (hazard ratio [HR]:1.96, p = 0.027). Contradicting published results, high hsa-miR-516-3p expression was associated with favorable outcome (HR:0.29, p = 0.0068). The association is probably time-dependent indicating later relapse. Additionally, re-analysis of previously published expression data of two matching cohorts (n = 100, n = 255) supports an association of hsa-miR-128-3p with shortened disease-free survival (HR:2.48, p = 0.0033) and an upregulation of miR-7-5p (p = 0.0038; p = 0.039) and miR-210-3p (p = 0.031) in primary tumors of patients who experienced metastases. Further analysis may verify the prognostic potential of these microRNAs.

Identification and expression analysis of microRNAs in medaka gonads

Gonad development is a highly regulated, coordinated biological process and increasing evidences have indicated that microRNA (miRNA) may be involved in this dynamic program. Medaka (Oryzias latipes) is a good model for reproductive research as it has distinct sex determining genes, however, research in gonadal miRNAs is lacked. In this study, two small RNA libraries from the ovaries and testes were constructed and sequenced. A total of 285 conserved and 388 novel miRNAs were obtained, among which 142 mature miRNAs were significantly (> two-fold change) up or down regulated in the testis compared to the ovary. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis showed that miR-430c, miR-26a and miR-202-5p were expressed in a gonad-specific or sex-biased pattern. Fluorescence in situ hybridization (FISH) indicated that miR-202-5p was present throughout spermatogenesis and was only detected at the early stages of oogenesis, this sex biased expression pattern suggested that miR-202-5p might be a crucial candidate in male differentiation and development. Our study provides the repertoire, a comprehensive annotation of miRNAs from gonads and a reference for functional studies of miRNAs in medaka.

Analysis of DNA methylation and microRNA expression in NUT (nuclear protein in testis) midline carcinoma of the sinonasal tract: a clinicopathological, immunohistochemical and molecular genetic study.

The aim of this study was a detailed clinicopathological investigation of sinonasal NUT midline carcinoma (NMC), including analysis of DNA methylation and microRNA (miRNA) expression. Three (5%) cases of NMC were detected among 56 sinonasal carcinomas using immunohistochemical screening and confirmed by fluorescence in situ hybridization. The series comprised 2 males and 1 female, aged 46, 60, and 65 years. Two tumors arose in the nasal cavity and one in the maxillary sinus. The neoplasms were staged pT1, pT3, and pT4a (all cN0M0). All patients were treated by radical resection with adjuvant radiotherapy. Two patients died 3 and 8 months after operation, but one patient (pT1 stage; R0 resection) experienced no evidence of disease at 108 months. Microscopically, all tumors consisted of infiltrating nests of polygonal cells with vesicular nuclei, prominent nucleoli and basophilic cytoplasm. Abrupt keratinization was present in only one case. Immunohistochemically, there was a diffuse expression of cytokeratin (CK) cocktail, CK7, p40, p63, and SMARCB1/INI1. All NMCs tested negative for EBV and HPV infection. Two NMCs showed methylation of RASSF1 gene. All other genes (APC, ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDH13, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, TIMP3, and VHL) were unmethylated. All NMCs showed upregulation of miR-9 and downregulation of miR-99a and miR-145 and two cases featured also upregulation of miR-21, miR-143, and miR-484. In summary, we described three cases of sinonasal NMCs with novel findings on DNA methylation and miRNA expression, which might be important for new therapeutic strategies in the future.

Analysis of Micro-RNA Expression by qPCR on a Microfluidics Platform for Alzheimer's Disease Biomarker Discovery.

Changes associated with neurodegeneration at the cellular level are manifestations of deregulated biochemical pathways, and typically precede neuronal loss. Incorporation of molecular markers in the diagnostic process could aid detection of early changes, prior to extensive neuronal loss, as early as the presymptomatic stages of the disorder, thus enabling improved patient stratification for targeted drug development. Such biomarkers should be sufficiently sensitive and specific to distinguish AD from other disorders with overlapping symptoms. Easily accessible biosamples, simple methodology, and low overall cost would enable population screening, which would not be feasible with other modalities. Non-coding (nc)RNAs have a crucial role in the entire spectrum of cellular processes, from development and differentiation to homeostatic maintenance, and have been implicated in different diseases; micro-RNAs (miRNAs) are a family of ncRNA molecules with an important role in posttranscriptional gene silencing. The early advances in the study of miRNAs as noninvasive biomarkers in cancer inspired their study for other conditions, including AD. Several deregulated miRNAs in brain, CSF, and blood have been associated with AD and other brain disorders. Their high stability makes miRNAs attractive for biomarker development, and a number of platforms are currently available for their analysis. qPCR is a technology characterized by high sensitivity and is suitable for focused analysis of specific candidates (assays) in a large number of samples. Microfluidic-based qPCR platforms have minimal RNA requirements and can yield thousands of datapoints in one qPCR experiment. Here, I present the use of miScript qPCR miRNA assays with the Fluidigm BioMark HD System.

Release of MicroRNAs into Body Fluids from Ten Organs of Mice Exposed to Cigarette Smoke.

Purpose: MicroRNAs are small non-coding RNAs that regulate gene expression, thereby playing a role in a variety of physiological and pathophysiological states. Exposure to cigarette smoke extensively downregulates microRNA expression in pulmonary cells of mice, rats, and humans. Cellular microRNAs are released into body fluids, but a poor parallelism was previously observed between lung microRNAs and circulating microRNAs. The purpose of the present study was to validate the application of this epigenetic biomarker by using less invasive collection procedures.Experimental design: Using microarray analyses, we measured 1135 microRNAs in 10 organs and 3 body fluids of mice that were either unexposed or exposed to mainstream cigarette smoke for up to 8 weeks. The results obtained with selected miRNAs were validated by qPCR.Results: The lung was the main target affected by smoke (190 dysregulated miRNAs), followed by skeletal muscle (180), liver (138), blood serum (109), kidney (96), spleen (89), stomach (36), heart (33), bronchoalveolar lavage fluid (32), urine (27), urinary bladder (12), colon (5), and brain (0). Skeletal muscle, kidney, and lung were the most important sources of smoke-altered microRNAs in blood serum, urine, and bronchoalveolar lavage fluid, respectively.Conclusions: microRNA expression analysis was able to identify target organs after just 8 weeks of exposure to smoke, well before the occurrence of any detectable histopathological alteration. The present translational study validates the use of body fluid microRNAs as biomarkers applicable to human biomonitoring for mechanistic studies, diagnostic purposes, preventive medicine, and therapeutic strategies.

Analysis of serum microRNA expression in male workers with occupational noise-induced hearing loss.

Occupational noise-induced hearing loss (ONIHL) is a prevalent occupational disorder that impairs auditory function in workers exposed to prolonged noise. However, serum microRNA expression in ONIHL subjects has not yet been studied. We aimed to compare the serum microRNA expression profiles in male workers of ONIHL subjects and controls. MicroRNA microarray analysis revealed that four serum microRNAs were differentially expressed between controls (n=3) and ONIHL subjects (n=3). Among these microRNAs, three were upregulated (hsa-miR-3162-5p, hsa-miR-4484, hsa-miR-1229-5p) and one was downregulated (hsa-miR-4652-3p) in the ONIHL group (fold change >1.5 and Pbon value <0.05). Real time quantitative PCR was conducted for validation of the microRNA expression. Significantly increased serum levels of miR-1229-5p were found in ONIHL subjects compared to controls (n=10 for each group; P<0.05). A total of 659 (27.0%) genes were predicted as the target genes of miR-1229-5p. These genes were involved in various pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of miR-1229-5p dramatically inhibited the luciferase activity of 3′ UTR segment of MAPK1 (P<0.01). Compared to the negative control, HEK293T cells expressing miR-1229-5p mimics showed a significant decline in mRNA levels of MAPK1 (P<0.05). This preliminary study indicated that serum miR-1229-5p was significantly elevated in ONIHL subjects. Increased miR-1229-5p may participate in the pathogenesis of ONIHL through repressing MAPK1 signaling.

MicroRNA Analysis Using Next-Generation Sequencing.

Next-generation sequencing (NGS) enables the analysis of both microRNA expression and sequence, allowing for elucidation of a comprehensive landscape of miRNAs in a given tissue and sample type. NGS analysis requires high-quality RNA extraction and preparation of microRNA libraries. In this chapter, we describe the methods used for RNA extraction from tissue specimens, serum, cytological slides, and formalin-fixed paraffin-embedded samples. Although the described library preparation and sequencing approaches are based on Illumina NextSeq 500 sequencing technology, the presented principles shall be compatible with other commercially available sequencing platforms.

Prediction of acute gastrointestinal and genitourinary radiation toxicity in prostate cancer patients using lymphocyte microRNA.

To search for novel biomarkers that can predict acute radiation toxicity, we conducted microRNA expression analysis of peripheral blood lymphocytes (PBLs). The discovery cohort was 69 patients with localized adenocarcinoma of the prostate who received intensity-modulated radiation therapy between October 2007 and October 2010. The validation cohort was 72 patients treated with low-dose-rate brachytherapy between May 2008 and March 2014. After13 microRNAs were selected by TaqMan® Array analysis in a preliminary experiment, expression of these microRNAs in all samples was analyzed by RT-PCR. In the discovery cohort, the average prostate volume, the rectal volume receiving 70 Gy, and expression of miR-410 and miR-221 were significant risk factors for Grade 1-2 gastrointestinal toxicity. Receiver operating characteristic analysis showed that the area under the curve (AUC) was 0.807. The maximum dose to the urinary bladder, prostate volume, pretreatment urinary function score, and miR-99a and miR-221 expression were risk factors for Grade 2 genitourinary toxicity. The corresponding AUC was 0.796. In the validation cohort, reproducibility of these markers was confirmed for gastrointestinal toxicity, but not for genitourinary toxicity. Combining radiation dose parameters with microRNA expression in PBLs may be useful for predicting acute gastrointestinal toxicity of radiation therapy, thus contributing to personalized treatment of prostate cancer.