Candidate microRNAs Research Articles

MiR-361 enhances sensitivity to 5-fluorouracil by targeting the FOXM1-ABCC5/10 signaling pathway in colorectal cancer.

Colorectal cancer (CRC) is one of most common malignancies worldwide. 5-fluorouracil (5-FU) is a mainstay of CRC treatment, particularly in patients with advanced stages of the disease; however, 5-FU-based chemotherapy is not always effective and may result in progression of the disease. The present study investigated several candidate microRNAs (miRs) in parental and 5-FU-resistant HCT116 and HT29 cells, and identified miR-361 as a novel regulator of chemosensitivity. Overexpression of miR-361 enhanced the 5-FU susceptibility of parental and resistant HCT116 and HT29 cells in vitro. Impaired colony formation capacity and increased cell apoptosis (as determined via flow cytometry) was observed in resistant HCT116 and HT29 cells. Furthermore, forkhead box M1 (FOXM1) was identified as a target gene of miR-361 using a dual-luciferase reporter assay, western blotting and reverse transcription-quantitative PCR. Additionally, FOXM1 knockdown improved the cytotoxicity of 5-FU in resistant CRC. ATP binding cassette subfamily C members 5 and 10 (ABCC5/10) were found to be downstream effectors of miR-361. In conclusion, miR-361 increased chemosensitivity, at least in part, via modulation of FOXM1-ABCC5/10. miR-361 may serve as a potential therapeutic target for patients with CRC.

Normal expression of KCNJ11 is maintained by the G-quadruplex

MicroRNAs affect various pathological pathways by binding to multiple target mRNAs. Recently, it was reported that eukaryotic RNA G-quadruplexes were enriched in mRNA 3′-UTR, where microRNAs can also bind. To determine the roles of the G-quadruplex within mRNA 3′-UTR in microRNA binding sites, a bioinformatics approach was utilized to identify candidate microRNA target mRNAs with potential G-quadruplex formation. Circular dichroism spectrometry demonstrated the formation of RNA G-quadruplexes in vitro in candidate G-rich sequences. Mutated guanosines that are critical for G-quadruplex formation significantly decreased luciferase activities. Moreover, a G-quadruplex ligand TMPyP4 was used to destabilize the KCNJ11 RNA G-quadruplex in cardiomyocytes, resulting in binding of the microRNA to mRNA and subsequent suppression of KCNJ11 expression. In conclusion, our study showed that the G-quadruplex structure affects microRNA binding to its target mRNA. Thus, our study reveals a novel mechanism for G-quadruplex-dependent regulation of microRNA-mRNA interaction, which is essential to maintain normal gene expression.

Long noncoding RNA SNHG12 indicates the prognosis of prostate cancer and accelerates tumorigenesis via sponging miR‐133b

Prostate cancer (PCa) isthe second leading cause of death among American men. Increasing evidence has shown that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis of PCa. In this study, we explored the biological functions of small nucleolar RNA host gene 12 (SNHG12) and investigated the interaction between miR-133b and SNHG12 in the progression of PCa. Data was downloaded from The Cancer Genome Atlas and Human Cancer Metastasis Database, and clinicopathological characteristics were analyzed with relapse-free survival rate. We detected SNHG12 expression level in PCa cells and tissues, and then analyzed its clinical significance, which revealed that SNHG12 has the potent to predict prognosis of PCa. Bioinformatic analysis revealed that SNHG12 was closely related to the progression of PCa and could target candidate microRNA (miR-133b). After transfecting SNHG12 silencing plasmid and miR-133b mimic/sponge, biological function assays were conducted and results illustrated that SNHG12 associated with miR-133b exerted biological effects on cancer cell growth, migration, and invasion. Direct interactions between miR-133b and SNHG12 have been found and SNHG12 acts as an oncogene to promote tumorigenesis of PCa by sponging tumor suppressor gene miR-133b.

Abstract 1800: miR-376b and miR-4668 predict therapeutic response to sunitinib in metastatic renal cell carcinoma

Abstract Introduction: Metastatic renal cell carcinoma (mRCC) is routinely treated with sunitinib, a tyrosine kinase inhibitor (TKI) of VEGF signalisation. Although disease eventually progresses in most mRCC patients, length of progression-free survival (PFS) is ranging from few weeks up to more than 24 months. Patients with initial resistance to sunitinib could be redirected to other therapeutical options, as there are several other TKIs available for use in second and third line. However, without a reliable biomarker no result can be predicted. MicroRNAs (miRNAs) belong to class of short non-coding RNAs and could serve as biomarkers of therapy response due to their unique character and presence in tissues and body fluids. Their biomarker potential has been discussed concerning many diseases including mRCC, but current knowledge is very weak, has several discrepancies and is acquired on relatively small cohorts. Material and method: Candidate microRNAs have been chosen based on global expression profiling using Affymetrix GeneChip 4.0 in 47 samples of FFPE mRCC tissue of patients treated with sunitinib (good response n=25, PFS longer than 17 months; poor response n=22, PFS shorter than 9 months). Validation was performed using qRT-PCR TaqMan assays on an independent cohort of 136 FFPE samples from mRCC patients treated with sunitinib with variable length of PFS. Local ethical committees at all involved centres approved the study protocol. The study was performed according to the World Medical Association Declaration of Helsinki and all patients signed an informed consent. Results and discussion: Of all tested miRNAs, expression of miR-4668-5p and miR-376b was the most significantly deregulated in non-responding patients with high statistical significance (p>0,005) and combined AUC higher than 0,8. Although other independent validations are necessary, microRNAs presented here seem to be very promising as tools for therapy personalization. Conclusion: We have successfully validated miR-4668-5p and miR-376b to have predictive potential in estimation of therapeutical response to sunitinib. Further functional analyses could shed more light on their involvement in development of therapy resistance. This work was supported by Ministry of Health of the Czech Republic, grant nr. NV18-03-00554. All rights reserved. Citation Format: Julia Kovacova, Jaroslav Juracek, Alexandr Poprach, Tomas Buchler, Ondrej Fiala, Lenka Radova, Marek Svoboda, Alena Kopkova, Marek Vecera, Ondrej Slaby. miR-376b and miR-4668 predict therapeutic response to sunitinib in metastatic renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1800.

MiR-146a and miR-181a are involved in the progression of mild cognitive impairment to Alzheimer's disease

The identification of mechanisms associated with Alzheimer's disease (AD) development in mild cognitive impairment (MCI) would be of great usefulness to clarify AD pathogenesis and to develop preventive and therapeutic strategies. In this study, blood levels of the candidate microRNAs (small noncoding RNAs that play a pivotal role in gene expression) miR-146a, miR-181a, miR-181b, miR-24-3p, miR-186a, miR-101, miR-339, miR-590, and miR-22 have been investigated for association to AD conversion within 2 years in a group of 45 patients with MCI. Baseline miR-146a (p = 0.036) and miR-181a (p = 0.026) showed a significant upregulation in patients with MCI who later converted to AD. These alterations were related to AD hallmarks: a significant negative correlation was found with amyloid beta cerebrospinal fluid concentration for miR-146a (p = 0.006) and miR-181a (p = 0.001). Moreover, higher levels of miR-146a were associated to apolipoprotein E ε4 allele presence, smaller volume of the hippocampus (p = 0.045) and of the CA1 (p = 0.013) and the subiculum (p = 0.027) subfields. Increased levels of miR-146a (p = 0.031) and miR-181a (p = 0.002) were also linked with diffusivity alterations in the cingulum. These data support a role for miR-146a and miR-181a in the mechanisms of AD progression.

Differentially expressed mRNAs, proteins and miRNAs associated to energy metabolism in skeletal muscle of beef cattle identified for low and high residual feed intake

BackgroundFeed efficiency is one of the most important parameters that affect beef production costs. The energy metabolism of skeletal muscle greatly contributes to variations in feed efficiency. However, information regarding differences in proteins involved in the energy metabolism of the skeletal muscle in beef cattle divergently identified for feed efficiency is scarce. In this study, we aimed to investigate energy metabolism of skeletal muscle of Nellore beef cattle, identified for low and high residual feed intake using a proteomics approach. We further assessed the expression of candidate microRNAs as a one of the possible mechanisms controlling the biosynthesis of the proteins involved in energy metabolism that were differentially abundant between high and low residual feed intake animals.ResultsA greater abundance of 14–3-3 protein epsilon (P = 0.01) was observed in skeletal muscle of residual feed intake (RFI) high animals (RFI-High). Conversely, a greater abundance of Heat Shock Protein Beta 1 (P < 0.01) was observed in the skeletal muscle of RFI-Low cattle. A greater mRNA expression of YWHAE, which encodes the 14–3-3 protein epsilon, was also observed in the skeletal muscle of RFI-High animals (P = 0.01). A lower mRNA expression of HSPB1, which encodes the Heat Shock Protein Beta 1, was observed in the skeletal muscle of RFI-High animals (P = 0.01). The miR-665 was identified as a potential regulator of the 14–3-3 protein epsilon, and its expression was greater in RFI-Low animals (P < .001). A greater expression of miR-34a (P = 0.01) and miR-2899 (P < .001) was observed in the skeletal muscle of RFI-High animals, as both miRNAs were identified as potential regulators of HSPB1 expression.ConclusionOur results show that Nellore cattle divergently identified for feed efficiency by RFI present changes in the abundance of proteins involved in energy expenditure in skeletal muscle. Moreover, our data point towards that miR-665, miR34a and miR-2899 are likely involved in controlling both 14-3-3 epsilon and HSPB1 proteins identified as differentially abundant in the skeletal muscle of RFI-High and RFI-Low Nellore cattle.

Trichloroethylene-induced downregulation of miR-199b-5p contributes to SET-mediated apoptosis in hepatocytes.

Trichloroethylene (TCE) is a ubiquitous toxicant widespread in our environment. Exposure to TCE can cause severe liver damage. In previous studies, we detected an abnormal elevation of SET (a protein encoded by the SETgene in humans) which was observed in human HL-7702 cells (L-02 hepatocytes) under the treatment of TCE. Moreover, further study indicated that SET acts as an important mediator in TCE-induced hepatocyte apoptosis. The major functions of SET have been elucidated, while the regulatory mechanism responsible for modulation of SET remains unclear. In this study, four major microRNA-related databases were used to screen and identify 6 candidate microRNAs involved in the regulation of SET. Subsequent verification indicated that miR-21 and miR-199b-5p were decreased in TCE-treated L-02 cells, suggesting that miR-21 and miR-199b-5p (miR199b for short) miR199b potentially regulate SET expression. Additionally, the dual-luciferase system revealed that only miR199b could directly bind to untranslated region (3'-UTR) of the SETgene. Modulation of SET by miR199b was verified through overexpression and knockdown of miR199b in L-02 cells. Assessment of apoptosis indicated that elevated miR199b attenuated TCE-induced apoptosis, while reduced miR199b enhanced it. In summary, this study suggests that in cultured hepatocytes, TCE-induced suppression of miR199b drives SET induction, which further mediates the response to TCE.

Genetic regulation of broodiness in poultry

Broodiness is a behavior commonly occurring in the poultry industry, which is characterized by inappetence, egg-laying cessation and incubation. Different from laying fowls, the ovary and oviduct of broodiness fowls is degenerate. Broodiness is a low heritability trait, which is controlled by multiple genes in autosomes and influenced by three factors, including environment, endocrine and genetics. In addition to the observation of behavioral characteristics, the current research of broodiness focuses on evaluating the genetic mode, endocrine factors, nesting candidate genes and their polymorphisms in poultry. Given the rapid development of high-throughput sequencing, the joint analyses of genomics, transcriptomics and proteomics have been used to screen out the candidate genes, pathways and molecular mechanism of broodiness. In this review, we summarize the candidate genes, microRNAs and pathways involved in broodiness to provide a reference for further research on poultry broodiness.

MicroRNA Biomarkers in Cerebrospinal Fluid and Serum Reflect Injury Severity in Human Acute Traumatic Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition with variability in injury mechanisms and neurologic recovery. Spinal cord impairment after SCI is measured and classified by a widely accepted standard neurological examination. In the very acute stages post-injury, however, this examination is extremely challenging (and often impossible) to conduct and has modest prognostic value in terms of neurological recovery. The lack of objective tools to classify injury severity and predict outcome is a barrier for clinical trials and thwarts development of therapies for those with SCI. Biological markers (biomarkers) represent a promising, complementary approach to these challenges because they represent an unbiased approach to classify injury severity and predict neurological outcome. Identification of a suitable panel of molecular biomarkers would comprise a fundamental shift in how patients with acute SCI are evaluated, stratified, and treated in clinical trials. MicroRNA are attractive biomarker candidates in neurological disorders for several reasons, including their stability in biological fluids, their conservation between humans and model mammals, and their tissue specificity. In this study, we used next-generation sequencing to identify microRNA associated with injury severity within the cerebrospinal fluid (CSF) and serum of human patients with acute SCI. The CSF and serum samples were obtained 1-5 days post-injury from 39 patients with acute SCI (24 American Spinal Injury Association Impairment Scale [AIS] A, 8 AIS B, 7 AIS C) and from five non-SCI controls. We identified a severity-dependent pattern of change in microRNA expression in CSF and identified a set of microRNA that are diagnostic of baseline AIS classification and prognostic of neurological outcome six months post-injury. The data presented here provide a comprehensive description of the CSF and serum microRNA expression changes that occur after acute human SCI. This data set reveals microRNA candidates that warrant further evaluation as biomarkers of injury severity after SCI and as key regulators in other neurological disorders.

Stage-dependent involvement of ADAM10 and its significance in epileptic seizures.

The prevalence of epileptic seizures in Alzheimer's disease (AD) has attracted an increasing amount of attention in recent years, and many cohort studies have found several risk factors associated with the genesis of seizures in AD. Among these factors, young age and severe dementia are seemingly contradictory and independent risk factors, indicating that the pathogenesis of epileptic seizures is, to a certain extent, stage‐dependent. A disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) is a crucial α‐secretase responsible for ectodomain shedding of its substrates; thus, the function of this protein depends on the biological effects of its substrates. Intriguingly, transgenic models have demonstrated ADAM10 to be associated with epilepsy. Based on the biological effects of its substrates, the potential pathogenic roles of ADAM10 in epileptic seizures can be classified into amyloidogenic processes in the ageing stage and cortical dysplasia in the developmental stage. Therefore, ADAM10 is reviewed here as a stage‐dependent modulator in the pathogenesis of epilepsy. Current data regarding ADAM10 in epileptic seizures were collected and reviewed for potential pathogenic roles (ie amyloidogenic processes and cortical dysplasia) and regulatory mechanisms (ie transcriptional and posttranscriptional regulation). These findings are then discussed in terms of the significance of the stage‐dependent functions of ADAM10 in epilepsy. Several potential targets for seizure control, such as candidate transcription factors and microRNAs that regulate ADAM10, as well as potential genetic screening tools for the early recognition of cortical dysplasia, have been suggested but must be studied in more detail.

MiR-31-5p Is a Potential Circulating Biomarker and Therapeutic Target for Oral Cancer

MicroRNAs have been proposed as novel biomarkers for the diagnosis and treatment of many types of cancer. The levels of five candidate microRNAs (miRNAs) (miR-99a-5p, miR-31-5p, miR-138-5p, miR-21-5p, and miR-375-3p) in sera from oral cancer patients and paired tumor and normal tissues were detected by real-time qPCR. The diagnostic power of these miRNAs was analyzed by receiver operating characteristic (ROC) curves. Patient-derived xenograft (PDX) models of oral cancer were established and utilized to verify the potential therapeutic effect of miR-31-5p. Candidate miRNAs were screened from our previous studies and verified in 11 paired oral cancer and adjacent normal tissues. Only serum miR-31-5p levels were significantly different between oral cancer patients and healthy controls and between pre- and postoperative patients. Based on the logistic regression model, this panel of five miRNAs distinguished oral cancer patients from healthy control, with an area under the ROC curve (AUC) of 0.776 (sensitivity = 76.8% and specificity = 73.6%). Furthermore, a miR-31-5p mimic enhanced the proliferation of normal epithelial cells, and antagomiR-31-5p inhibited the proliferation of oral cancer cells in vitro. In vivo, antagomiR-31-5p significantly inhibited tumor growth in oral cancer PDX models. Our findings suggest that circulating miR-31-5p might act as an independent biomarker for oral cancer diagnosis and could serve as a therapeutic target for oral cancer.

Screening of differentially expressed microRNAs of essential hypertension in Uyghur population

BackgroundEssential hypertension can cause many kinds of cardiovascular diseases. The pathogenesis of essential hypertension is very complex, and the mechanism is still unclear. The microRNAs have been identified as novel biomarkers for pre-diagnosis and prognosis of hypertension. However, the kinds of microRNAs that can be used as specific biomarkers for hypertension are unknown.Methods and resultsPlasma samples were isolated from Uyghur subjects with essential hypertension and the healthy individuals. Microarray was used to identify differentially expressed microRNAs. The microarray data were clustered and annotated with online software. The target genes of differentially expressed microRNAs were also analyzed. The microarray results were further verified by quantitative real-time PCR. We identified 257 microRNAs that were differentially expressed between patients with essential hypertension and the healthy individuals. These microRNAs had a total of 6580 target genes. The 47 microRNAs that had target genes, including 24 up-regulated and 23 down-regulated microRNAs, were further screened out to construct a reference set of potential microRNA biomarkers. Most of the 47 microRNAs were located at chromosome 19 (40 microRNAs) and chromosome 1 (45 microRNAs). Their target genes were mainly enriched in metal ion binding, transcription regulation, cell adhesion and junction, indicating that these candidate microRNAs may regulate mineral ion binding and cell communication process of essential hypertension. The quantitative real-time PCR results of miR-198 and miR-1183 (which were the two most significantly up-regulated microRNAs by microarray), and, miR-30e-5p and miR-144-3p (which were the two most significantly down-regulated microRNAs by microarray) were consistent with the microarray results.ConclusionsA reference set of potential microRNA biomarkers that may be involved in essential hypertension is constructed. Our study may provide experimental evidence for further studying the mechanism of essential hypertension.

Down-regulation of miRNA-27b-3p suppresses keratinocytes apoptosis in oral lichen planus.

Oral lichen planus (OLP) is considered a precancerous lesion with no known cure. Recent studies reported that abnormal regulation of apoptosis was involved in the pathogenesis of OLP. Next generation sequencing was used to screen the candidate microRNAs and genes in biopsies from patients with OLP and healthy mucosa. Human oral keratinocytes were transfected into the related oligonucleotides of miR‐27b‐3p/cyclophilin D and their control groups. Apoptosis was detected by TdT‐mediated dUTP nick end labelling and flow cytometry. The levels of mRNA and protein were detected by quantitative PCR, Western blots, and enzyme‐linked immunosorbent assays, respectively. Luciferase assays were performed to detect the luciferase activities of miR‐27b‐3p and cyclophilin D. Here, we showed that basal epithelium apoptosis was reduced and the miR‐27b‐3p levels were decreased in clinical OLP samples. We also found that down‐regulation of miR‐27b‐3p inhibited epithelial keratinocyte apoptosis by up‐regulating cyclophilin D expression. Moreover, cyclophilin D increased the protein stability of Bcl2 through direct binding, and Bcl2 suppressed caspase9/3 activation and cytochrome C release. Taken together, these data showed that miR‐27b‐3p regulated keratinocyte apoptosis through cyclophilin D/Bcl2 signalling, suggesting the miR‐27b‐3p regulated the pathogenesis of OLP.

Sex Differences in Plasma MicroRNA Biomarkers of Early and Complicated Diabetes Mellitus in Israeli Arab and Jewish Patients.

MicroRNAs play functional roles in the etiology of type 2 diabetes mellitus (T2DM) and complications, and extracellular microRNAs have attracted interest as potential biomarkers of these conditions. We aimed to identify a set of plasma microRNAs, which could serve as biomarkers of T2DM and complications in a mixed Israeli Arab/Jewish patient sample. Subjects included 30 healthy volunteers, 29 early-stage T2DM patients, and 29 late-stage T2DM patients with renal and/or vascular complications. RNA was isolated from plasma, and the levels of 12 candidate microRNAs were measured by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). MicroRNA levels were compared between the groups and correlated to clinical measurements, followed by stepwise regression analysis and discriminant analysis. Plasma miR-486-3p and miR-423 were respectively up- and down-regulated in T2DM patients compared to healthy controls. MiR-28-3p and miR-423 were up-regulated in patients with complicated T2DM compared to early T2DM, while miR-486-3p was down-regulated. Combined, four microRNAs (miR-146a-5p, miR-16-2-3p, miR-126-5p, and miR-30d) could distinguish early from complicated T2DM with 77% accuracy and 79% sensitivity. In male patients only, the same microRNAs, with the addition of miR-423, could distinguish early from complicated T2DM with 83.3% accuracy. Furthermore, plasma microRNA levels showed significant correlations with clinical measurements, and these differed between men and women. Additionally, miR-183-5p levels differed significantly between the ethnic groups. Our study identified a panel of specific plasma microRNAs which can serve as biomarkers of T2DM and its complications and emphasizes the importance of sex differences in their clinical application.

Expression levels of miR-143-3p and -424-5p in colorectal cancer and their clinical significance.

Colorectal cancer (CRC) is one of the most prevalent cancers and microRNAs are involved in colorectal carcinogenesis and progression. The role of our candidate microRNAs (miR-143-3p, -424-5p, -212-3p and -34a-3p) have been investigated in various cancers. The aim of the current study was to evaluate expression levels of microRNAs (miR-143-3p, -424-5p, -212-3p and -34a-3p) in the sera of patients with CRC in order to identify potential non-invasive biomarker for CRC and investigate the relationship between their expression and clinicopathological features of CRC. The serological expression of candidate microRNAs were measured in 124 participants, including 62 CRC patients and 62 healthy controls and the serum expression levels of candidate miRNAs were quantified by stemloop reverse transcriptionquantitative polymerase chain reaction. In the present study, results showed a significant upregulation expression level of miR-424-5p (P< 0.001) and decreased expression level of miR143-3p was observed in the sera of patients with CRC (P< 0.001). Receiver operating characteristic (ROC) curve analysis demonstrated the area of miR-424-5p and miR-143-3p under the ROC curve for CRC diagnosis were 0.703 and 0.724 respectively (P< 0.001). In addition, down expression of miR-143-3p was significantly associated with tumor size (P= 0.005) and lymph node metastasis (P= 0.020) in CRC patients. The present investigation suggested that low expression of miR-143-3p and increasing level of miR-424-5p in CRC patients may play an important role in development of CRC and they could function as potential non-invasive biomarkers for CRC.

Circulating microRNA signatures identified in early versus late knee osteoarthritis

Purpose: There continues to be a lack of specific osteoarthritis (OA) biomarkers that can be used in a therapeutic, prognostic, or diagnostic manner. Since microRNAs are promising biomarkers for several diseases, we previously used microRNA PCR-arrays and identified a panel of microRNAs in the synovial fluid that differentiate between early- and late-stage radiographic knee OA. We also performed microRNA microarrays and identified 2 microRNAs as mediators of cartilage degeneration. However, the gold standard approach to identifying biomarkers is next generation sequencing because it offers the sensitivity and specificity to detect novel and low abundance microRNAs that are unique to various cohorts (early versus late OA, men versus women, young versus old, normal weight versus overweight). Here, we use next generation sequencing to identify signatures of circulating microRNAs as biomarkers for knee OA. We hypothesize that identification and characterization of known and novel circulating microRNAs can be used as biomarker signatures to define cohorts of patients with OA. Methods: Plasma samples from knee OA patients are obtained from the Knee Osteoarthritis BioBank at the University Health Network in Toronto, Canada. Samples include healthy donors with no history of musculoskeletal disease (N=100), Kellgren-Lawrence grades 0 and 1 for early OA (N=100), and Kellgren-Lawrence grades 3 and 4 for late OA (N=750). Patients with comorbidities that might affect microRNA signatures are excluded. In a pilot experiment, plasma samples from 5 healthy donors, 5 early OA patients, and 5 late OA patients were subjected to next generation sequencing of microRNAs. Differentially expressed microRNAs were identified using the negative binomial exact test. To validate our findings, we are currently sequencing plasma from larger cohorts of patients. Further statistical analysis will be used to identify relationships between microRNA signatures and known risk factors such as sex, age, and body mass index. Bioinformatic methods will be used to predict the targets and biological function of candidate microRNAs in OA. Results: After mapping sequencing reads to mirbase20, 164 microRNAs with greater than 10 counts per million were identified in all samples. Principal component analysis was performed using the 50 microRNAs with the largest coefficient of variation based on normalized counts. This revealed a clear separation of early OA samples from both late OA and healthy samples. The most differentially expressed microRNAs were identified based on false discovery rate less than 0.05, log counts per million greater than 2, and log fold change greater than 1.5. Hierarchical clustering of these microRNAs revealed a distinct pattern where 60 microRNAs were upregulated only in early OA samples (Figure 1). Among these are 4 novel putative microRNAs that have not previously been identified. We are currently validating these findings with additional sequencing experiments in each group (N=100 healthy donors, N=100 early OA, N=750 late OA) to establish a clear microRNA signature for early knee OA. Conclusions: Data from our pilot experiment demonstrate that next generation sequencing is a useful approach for identifying known and novel circulating microRNAs in OA. Sequencing of early OA samples will allow identification of microRNA signatures that can be used to distinguish these patients from late OA patients. Circulating microRNAs may represent valid and reliable biomarkers with potential applications for improving OA detection and treatment.

MicroRNA-431 as a Chemosensitizer and Potentiator of Drug Activity in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare endocrine cancer with treatments limited in efficacy for metastatic disease. New molecular targeted therapies have yet to improve patient outcomes. In contrast, established treatment regimens of adrenolytics and chemotherapy have demonstrated treatment benefit, although admittedly in a minority of patients. Identification of microRNAs (miRNAs) in patients responsive to adjuvant therapy may offer a means to sensitize patients with progressive disease to existing adjuvant regimens. Samples from primary ACC tumors of 10 Stage IV patients were examined for differentially expressed miRNAs between a "sensitive" and "resistant" cohort. Candidate microRNAs were restored via transfection in two functional ACC cell lines. Gain of function and effects on apoptosis and cell cycle were assessed. microRNA-431 (miR-431) was underexpressed in patients with ACC with progressive disease undergoing adjuvant therapy. Restoration of miR-431 in vitro decreased the half maximal inhibitory concentrations of doxorubicin and mitotane, with markedly increased apoptosis. We found that a reversal of epithelial-mesenchymal transition underlies the action of miR-431 with doxorubicin treatment, with Zinc Finger E-Box Binding Homeobox 1 implicated as the molecular target of miR-431 in ACC. This is the first report of the potential of miRNA therapy to sensitize ACC to current established adjuvant therapy regimens, which may mitigate the resistance underlying treatment failure in patients with advanced ACC. Effective and well-studied methods of targeted miRNA delivery in existence hints at the imminent translatability of these findings. Adrenocortical carcinoma (ACC) is a rare endocrine cancer with outcomes not improving despite extensive research and new targeted therapies. Mitotane and etoposide/doxorubicin/cisplatin chemotherapy is trial validated for improved recurrence-free survival. However, a minority of patients experience sustained benefit. Significant side effects exist for this regimen, with patients often unable to attain target drug doses shown to give survival benefit. This preclinical study examines the role of microRNAs in sensitizing ACC to doxorubicin or mitotane. This study offers an important bridge between new and existing cancer treatments, offering an imminently translatable approach to the treatment of adrenocortical carcinoma.

Identification of Candidate Genes and MicroRNAs for Acute Myocardial Infarction by Weighted Gene Coexpression Network Analysis.

Background Identification of potential molecular targets of acute myocardial infarction is crucial to our comprehensive understanding of the disease mechanism. However, studies of gene coexpression analysis via jointing multiple microarray data of acute myocardial infarction still remain restricted. Methods Microarray data of acute myocardial infarction (GSE48060, GSE66360, GSE97320, and GSE19339) were downloaded from Gene Expression Omnibus database. Three data sets without heterogeneity (GSE48060, GSE66360, and GSE97320) were subjected to differential expression analysis using MetaDE package. Differentially expressed genes having upper 25% variation across samples were imported in weighted gene coexpression network analysis. Functional and pathway enrichment analyses were conducted for genes in the most significant module using DAVID. The predicted microRNAs to regulate target genes in the most significant module were identified using TargetScan. Moreover, subpathway analyses using iSubpathwayMiner package and GenCLiP 2.0 were performed on hub genes with high connective weight in the most significant module. Results A total of 1027 differentially expressed genes and 33 specific modules were screened out between acute myocardial infarction patients and control samples. Ficolin (collagen/fibrinogen domain containing) 1 (FCN1), CD14 molecule (CD14), S100 calcium binding protein A9 (S100A9), and mitochondrial aldehyde dehydrogenase 2 (ALDH2) were identified as critical target molecules; hsa-let-7d, hsa-let-7b, hsa-miR-124-3, and hsa-miR-9-1 were identified as potential regulators of the expression of the key genes in the two biggest modules. Conclusions FCN1, CD14, S100A9, ALDH2, hsa-let-7d, hsa-let-7b, hsa-miR-124-3, and hsa-miR-9-1 were identified as potential candidate regulators in acute myocardial infarction. These findings might provide new comprehension into the underlying molecular mechanism of disease.

Serum Circulating miR-150 is a Predictor of Post-Acute Myocardial Infarction Heart Failure.

Patients with ischemic heart disease are associated with poor prognosis, and their number has increased globally. Therefore, biomarkers that could predict post-acute myocardial infarction (AMI) heart failure (HF) would be helpful to guide appropriate treatment. Based on the diagnosis on admission and results of echocardiogram performed on admission and 1 year after discharge, the current study recruited 54 patients with post-AMI HF, 59 patients with post-AMI non-HF, and 59 healthy controls. Eight candidate microRNAs (miRs) were screened through real-time quantitative PCR. Serum circulating miR-150 level in the post-AMI HF group was significantly lower than the post-AMI non-HF group (0.4 ± 0.3 versus 0.7 ± 0.3, P < 0.001). Further analysis showed that serum circulating miR-150 level was associated with ejection fraction (EF) 1 year after discharge (P < 0.001). Receiver operating characteristic curve (ROC) analysis found that area under the ROC (AUC) was 0.616 (95%CI = 0.511-0.721, P = 0.034) when BNP was used to predict post-AMI HF, whereas AUC improved to 0.764 (95%CI = 0.674-0.855, P < 0.001) when miR-150 was used. The combination of BNP and miR-150 significantly improved the AUC to 0.807 (95%CI = 0.727-0.886, P < 0.001). Finally, multivariate logistic regression analysis revealed that either LVEF on admission or serum circulating miR-150 level was independently associated with post-AMI HF. Serum circulating miR-150 is a novel biomarker to predict post-AMI HF. Further large sample prospective clinical research is needed to validate its role in the future.

Human Novel MicroRNA Seq-915_x4024 in Keratinocytes Contributes to Skin Regeneration by Suppressing Scar Formation

Early in gestation, wounds in fetal skin heal by regeneration, in which microRNAs play key roles. Seq-915_x4024 is a novel microRNA candidate confirmed by deep sequencing and mirTools 2.0. It is highly expressed in fetal keratinocytes during early gestation. Using an in vitro wound-healing assay, Transwell cell migration assay, and MTS proliferation assay, we demonstrated that keratinocytes overexpressing seq-915_x4024 exhibited higher proliferative activity and the ability to promote fibroblast migration and fibroblast proliferation. These characteristics of keratinocytes are the same biological behaviors as those of fetal keratinocytes, which contribute to skin regeneration. In addition, seq-915_x4024 suppressed the expression of the pro-inflammatory markers TNF-α, IL-6, and IL-8 and the pro-inflammatory chemokines CXCL1 and CXCL5. We also demonstrated that seq-915_x4024 regulates TGF-β isoforms and the extracellular matrix. Moreover, using an in vivo wound-healing model, we demonstrated that overexpression of seq-915_x4024 in keratinocytes suppresses inflammatory cell infiltration and scar formation. Using bioinformatics analyses, luciferase reporter assays, and western blotting, we further demonstrated that Sar1A, Smad2, TNF-α, and IL-8 are direct targets of seq-915_x4024. Furthermore, the expression of phosphorylated Smad2 and Smad3 was reduced by seq-915_x4024. Seq-915_x4024 could be used as an anti-fibrotic factor for the treatment of wound healing.