Identification of Muscle-Specific MicroRNAs in Serum of Muscular Dystrophy Animal Models: Promising Novel Blood-Based Markers for Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein, dystrophin. Creatine kinase (CK) is generally used as a blood-based biomarker for muscular disease including DMD, but it is not always reliable since it is easily affected by stress to the body, such as exercise. Therefore, more reliable biomarkers of muscular dystrophy have long been desired. MicroRNAs (miRNAs) are small, ∼22 nucleotide, noncoding RNAs which play important roles in the regulation of gene expression at the post-transcriptional level. Recently, it has been reported that miRNAs exist in blood. In this study, we hypothesized that the expression levels of specific serum circulating miRNAs may be useful to monitor the pathological progression of muscular diseases, and therefore explored the possibility of these miRNAs as new biomarkers for muscular diseases. To confirm this hypothesis, we quantified the expression levels of miRNAs in serum of the dystrophin-deficient muscular dystrophy mouse model, mdx, and the canine X-linked muscular dystrophy in Japan dog model (CXMDJ), by real-time PCR. We found that the serum levels of several muscle-specific miRNAs (miR-1, miR-133a and miR-206) are increased in both mdx and CXMDJ. Interestingly, unlike CK levels, expression levels of these miRNAs in mdx serum are little influenced by exercise using treadmill. These results suggest that serum miRNAs are useful and reliable biomarkers for muscular dystrophy

Identification of circulating microRNAs as diagnostic biomarkers for use in multiple myeloma

BACKGROUND: Multiple myeloma is a plasma cell disorder that is characterised by clonal proliferation of malignant plasma cells in the bone marrow, monoclonal paraprotein in the blood or urine and associated organ dysfunction. It accounts for approximately 1% of cancers and 13% of haematological cancers. Myeloma arises from an asymptomatic proliferation of monoclonal plasma cells termed monoclonal gammopathy of undetermined significance (MGUS). METHODS: MicroRNA expression profiling of serum samples was performed on three patient groups as well as normal controls. Validation of the nine microRNAs detected as promising biomarkers was carried out using TaqMan quantitative RT-PCR. MicroRNA levels in serum were normalised using standard curves to determine the numbers of microRNAs per μl of serum. RESULTS: Three serum microRNAs, miR-720, miR-1308 and miRNA-1246, were found to have potential as diagnostic biomarkers in myeloma. Use of miR-720 and miR-1308 together provides a powerful diagnostic tool for distinguishing normal healthy controls, as well as patients with unrelated illnesses, from precancerous myeloma and myeloma patients. In addition, the combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients. CONCLUSION: We have developed a biomarker signature using microRNAs extracted from serum which has potential as a diagnostic and prognostic tool for multiple myeloma

A Pilot Study of Circulating miRNAs as Potential Biomarkers of Early Stage Breast Cancer

To date, there are no highly sensitive and specific minimally invasive biomarkers for detection of breast cancer at an early stage. The occurrence of circulating microRNAs (miRNAs) in blood components (including serum and plasma) has been repeatedly observed in cancer patients as well as healthy controls. Because of the significance of miRNA in carcinogenesis, circulating miRNAs in blood may be unique biomarkers for early and minimally invasive diagnosis of human cancers. The objective of this pilot study was to discover a panel of circulating miRNAs as potential novel breast cancer biomarkers.Using microarray-based expression profiling followed by Real-Time quantitative Polymerase Cycle Reaction (RT-qPCR) validation, we compared the levels of circulating miRNAs in plasma samples from 20 women with early stage breast cancer (10 Caucasian American (CA) and 10 African American (AA)) and 20 matched healthy controls (10 CAs and 10 AAs). Using the significance level of p<0.05 constrained by at least two-fold expression change as selection criteria, we found that 31 miRNAs were differentially expressed in CA study subjects (17 up and 14 down) and 18 miRNAs were differentially expressed in AA study subjects (9 up and 9 down). Interestingly, only 2 differentially expressed miRNAs overlapped between CA and AA study subjects. Using receiver operational curve (ROC) analysis, we show that not only up-regulated but also down-regulated miRNAs can discriminate patients with breast cancer from healthy controls with reasonable sensitivity and specificity. To further explore the potential roles of these circulating miRNAs in breast carcinogenesis, we applied pathway-based bioinformatics exploratory analysis and predicted a number of significantly enriched pathways which are predicted to be regulated by these circulating miRNAs, most of which are involved in critical cell functions, cancer development and progression.Our observations from this pilot study suggest that the altered levels of circulating miRNAs might have great potential to serve as novel, noninvasive biomarkers for early detection of breast cancer

Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format

Universal Oligonucleotide Microarray for Sub-Typing of Influenza A Virus

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1–H13, H15, H16) and neuraminidase (N1–N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus

Effects of mannoprotein E1 in liquid diet on inflammatory response and TLR5 expression in the gut of rats infected by Salmonella typhimurium

<p>Abstract</p> <p>Background</p> <p>Mannoproteins are yeast cell wall componend, and rich in mannose. The use of foods rich in mannose as carbohydrate, could have a bioprotective effect against entrobacteria intestinal infection. Nothing is known about mannoproteins' activity in inflammatory bowel processes induced by entrobacteria.</p> <p>This study investigates the effects of mannoprotein administration via a liquid diet on inflammatory response and TLR5 expression during intestinal tissue injury in a rat model of infection with <it>Salmonella typhimurium</it>.</p> <p>Methods</p> <p>Adult Wistar male rats were divided into three groups: control, and mannoprotein E₁at 10 or 15%. Animals were fed with a liquid diet supplemented or not with mannoprotein E₁. Groups were infected by intragastrical administration of <it>S. typhimurium</it>. 24 h post-inoculation samples of spleen, ileum and liver were collected for microbiological studies. Gut samples were processed to determine levels of proinflammatory cytokines (mRNA) and TLR5 (mRNA and protein) by quantitative PCR and Western-blot, and the number of proliferative and apoptotic cells determined by immunohistochemistry.</p> <p>Results</p> <p>Ininfected levels of proinflammatory cytokines and TLR5 were higher in untreated controls than in the animals receiving mannoprotein. Proliferation was similar in both groups, whereas apoptosis was higher in controls. Curiosly, the mannoprotein effect was dose dependent.</p> <p>Conclusions</p> <p>Mannoprotein administration in a liquid diet seems to protect intestinal tissue against <it>S. typhimurium </it>infection. This protection seems to expressed as a lower pro-inflammatory response and TLR5 downregulation in gut epithelium, as well as by an inhibition of apoptosis. Nevertheless, the molecular mechanism by which mannoprotein is able to regulate these responses remain unclear. These results could open up new avenues in the use of mannoproteins as prebiotics in the therapeutic strategy for treatment of inflammatory gut processes induced by microbia.</p

MicroRNA Profile Predicts Recurrence after Resection in Patients with Hepatocellular Carcinoma within the Milan Criteria

Objective: Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection. Methods: We examined microRNA expression profiling in paired tumor and non-tumor liver tissues from 73 HCC patients who satisfied the Milan Criteria. We constructed prediction models of recurrence-free survival using the Cox proportional hazard model and principal component analysis. The prediction efficiency was assessed by the leave-one-out crossvalidation method, and the time-averaged area under the ROC curve (ta-AUROC). Results: The univariate Cox analysis identified 13 and 56 recurrence-related microRNAs in the tumor and non-tumor tissues, such as miR-96. The number of recurrence-related microRNAs was significantly larger in the non-tumor-derived microRNAs (N-miRs) than in the tumor-derived microRNAs (T-miRs, P, 0.0001). The best ta-AUROC using the whole dataset, T-miRs, NmiRs, and clinicopathological dataset were 0.8281, 0.7530, 0.7152, and 0.6835, respectively. The recurrence-free survival curve of the low-risk group stratified by the best model was significantly better than that of the high-risk group (Log-rank: P = 0.00029). The T-miRs tend to predict early recurrence better than late recurrence, whereas N-miRs tend to predict late recurrence better (P, 0.0001). This finding supports the concept of early recurrence by the dissemination of primary tumor cells and multicentric late recurrence by the ‘field effect’. Conclusion: microRNA profiling can predict HCC recurrence in Milan criteria cases

Serum MicroRNA Expression Profile Distinguishes Enterovirus 71 and Coxsackievirus 16 Infections in Patients with Hand-Foot-and-Mouth Disease

Altered circulating microRNA (miRNA) profiles have been noted in patients with microbial infections. We compared host serum miRNA levels in patients with hand-foot-and-mouth disease (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) as well as in other microbial infections and in healthy individuals. Among 664 different miRNAs analyzed using a miRNA array, 102 were up-regulated and 26 were down-regulated in sera of patients with enteroviral infections. Expression levels of ten candidate miRNAs were further evaluated by quantitative real-time PCR assays. A receiver operating characteristic (ROC) curve analysis revealed that six miRNAs (miR-148a, miR-143, miR-324-3p, miR-628-3p, miR-140-5p, and miR-362-3p) were able to discriminate patients with enterovirus infections from healthy controls with area under curve (AUC) values ranged from 0.828 to 0.934. The combined six miRNA using multiple logistic regression analysis provided not only a sensitivity of 97.1% and a specificity of 92.7% but also a unique profile that differentiated enterovirial infections from other microbial infections. Expression levels of five miRNAs (miR-148a, miR-143, miR-324-3p, miR-545, and miR-140-5p) were significantly increased in patients with CVA16 versus those with EV71 (p<0.05). Combination of miR-545, miR-324-3p, and miR-143 possessed a moderate ability to discrimination between CVA16 and EV71 with an AUC value of 0.761. These data indicate that sera from patients with different subtypes of enteroviral infection express unique miRNA profiles. Serum miRNA expression profiles may provide supplemental biomarkers for diagnosing and subtyping enteroviral HFMD infections

Let-7 MicroRNA Family Is Selectively Secreted into the Extracellular Environment via Exosomes in a Metastatic Gastric Cancer Cell Line

Background: Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene downregulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. Methodology/Principal Findings: Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. Conclusions/Significance: The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosome