Presence Of miRs Research Articles

Dengue Virus Serotypes Circulating among Aedes Mosquitoes in the Lucknow District of North India: Molecular Identification and Characterization

Dengue is one of the most critical diseases, caused by Dengue virus (DENV) serotypes (DENV-1 to DENV-4). Study aims to detect DENV in natural A. aegypti from endemic regions of Uttar Pradesh. From 2010- 2013, mosquitoes collected from Uttar Pradesh tested for Dengue virus serotypes by capsid-premembrane gene-based PCR analyzed for genotypes sequencing of the C-prM junction of DENV genome. A total of 4731, 53.54% (n=2671) A. aegypti and 46 % (n=2060) A. albopictus mosquitoes were collected. Of 226 mosquito pools, 10 pools of A. aegypti and 14 pools of A. albopictus were positive for DENV by PCR. All 24 isolates identified as DENV-I; Genotype (G)-III (n=8), G-V (n=1); DENV-2; G-IV (n=4); DENV-3; G-III (n=11). The overall minimum infection rate was much higher in A. albopictus mosquitoes and presence of MIR in male mosquitoes is an indicating natural vertical transmission and important observation in geographical area indicating natural vertical transmission.

Serum miR-214 as a novel biomarker for ankylosing spondylitis.

Serum microRNA (miR) in ankylosing spondylitis (AS) patients has been rarely identified. The objective of this study was to find AS-specific miR in sera of patients with AS. Total RNAs were isolated from whole sera of patients with AS, patients with rheumatoid arthritis (RA), and healthy controls (HC) using miRNeasy Serum/Plasma Kit. The presence of miR was assayed using Agilent 2100 Bioanalyzer Small RNA assay. Each RNA sample was used for miR microarray. To verify microarray results, candidate circulating miRs were validated by quantitative polymerase chain reaction (qPCR) using samples from patients with AS (n=65), patients with RA (n=25), and HCs (n=39). Cycle threshold values were converted to copy numbers by drawing a standard curve using a synthetic chemical standard. All clinical values were also evaluated at the time of miR isolation. A total of 887 miRs were screened for three groups. Lower expression of miR-214 in AS than in HC and RA was observed after normalization of raw data. Finally, lower expression of serum miR-214 was confirmed in AS after validation by qPCR. Correlation analysis showed that the level of miR-214 of AS was significantly associated with Ankylosing Spondylitis Disease Activity Score-C-reactive protein (r=0.299, P=0.02). However, other disease-specific variables showed no statistical significance: gender (P=0.286), peripheral arthritis (P=0.634), enthesitis (P=0.464), dacylitis (P=0.750), psoriasis (P=0.552), inflammatory bowel disease (P=0.369), human leukocyte antigen-B27 positivity (P=0.473), use of non-steroidal anti-inflammatory drugs (P=0.448), and use of tumor necrosis factor-blocker in the last 3months (P=0.505). miR-214 may serve as a noninvasive biomarker for diagnosis of AS. In addition, expression level of miR-214 was associated with disease activity.

Abstract 3205: Breast cancer cell quiescence in bone is modulated by Osteo-TAFs

Abstract Breast cancer frequently metastasizes to bone. Upon entry to a secondary site, disseminated breast cancer cells (BCCs) can undergo a period of proliferative quiescence for ≤25 years where they remain clinically undetectable and untreatable. We propose that disseminated BCCs educate osteoblasts (OBs) into a tumor-associated fibroblast (TAF)-like cell that, in turn, orchestrates the proliferative quiescence BCC phenotype in bone. To test this, differentiated MC3T3-E1 murine osteoblasts (OBs) were incubated for 21 days with conditioned medium (CM) from metastatic BCC variants (MDA-231 or metastasis-suppressed MDA-231). OB cell lysates were analyzed for TAF proteins. Importantly, OBs reduced their expression of alkaline phosphatase, F4/80, CD31, and alpha SMA, with the largest change seen in 41-day-old conditioned OBs. These results are consistent with the described aggressive “matrix-remodeling” TAF. On the other hand, metastasis-suppressed BC CM treated OBs increased their expression of IL-6, suggestive of an OB inflammatory response to disseminated BCCs. TAF-OBs also exhibited altered morphology in the form of a thin, spindle-like shape, as opposed to cobblestone appearance. As a way to better understand how TAF-OBs can induce proliferative quiescence in BCC we sought to examine the roles of gap junctions and exchange of miRs as modulators to this process. Briefly, OBs were differentiated and ad-mixed with 1) GFP-MDA-231 human BCCs, or 2) GFP-MDA-231 BCCs double-labeled with CellTracker Orange CMTMR. Forty-eight hours later, these co-cultures were assessed for the presence of gap junction protein expression (Cx43, representative gap junction), and whether they functioned to exchange information using a cellular CMTMR dye transfer assay. Cx43 expression was localized specifically between OBs and BCCs. Interestingly, we observed direct cell-cell dye transfer occurring between BCCs that distributed dye to OBs several cell distances away. Next, we assayed supernatants of OBs and BCCs for the presence of exosomes and exosomal miRs, both known mediators of cell-to-cell communication. Both OBs and BCCs produced exosomes as characterized by transmission electron microscopy and a Nanosight 500 System. OB-derived exosomes were visualized in co-culture assays using a CD63-RFP immunolabel. Late differentiated OBs produced over twice the amount of exosomes than did OBs in their growth phase. Given this, we interrogated the contents of the exosomes for miRs that might induce proliferative quiescence in BCCs. miR arrays demonstrated the presence of miRs involved in cell cycle control (miR 193) and cellular dormancy (miR 320a) as produced by TAF-OBs. Combined, these data suggest that there is extensive crosstalk between OBs and disseminated BCCs in bone. This work highlights the importance of investigating OBs as key players in BCC dormancy in bone. Supported by NIH 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; (NRSA) T32 CA079448, NIH 1 K99 CA178177 for KMB. Citation Format: Karen M. Bussard, Frank C. Marini. Breast cancer cell quiescence in bone is modulated by Osteo-TAFs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3205. doi:10.1158/1538-7445.AM2015-3205

Nanofiber-mediated microRNA delivery to enhance differentiation and maturation of oligodendroglial precursor cells

Remyelination in the central nervous system (CNS) is critical in the treatment of many neural pathological conditions. Unfortunately, the ability to direct and enhance oligodendrocyte (OL) differentiation and maturation remains limited. It is known that microenvironmental signals, such as substrate topography and biochemical signaling, regulate cell fate commitment. Therefore, in this study, we developed a nanofiber-mediated microRNA (miR) delivery method to control oligodendroglial precursor cell (OPC) differentiation through a combination of fiber topography and gene silencing. Using poly(ε-caprolactone) nanofibers, efficient knockdown of OL differentiation inhibitory regulators were achieved by either nanofiber alone (20–40%, p<0.05) or the synergistic integration with miR-219 and miR-338 (up to 60%, p<0.05). As compared to two-dimensional culture, nanofiber topography enhanced OPC differentiation by inducing 2-fold increase in RIP+ cells (p<0.01) while the presence of miRs further enhanced the result to 3-fold (p<0.001). In addition, nanofiber-mediated delivery of miR-219 and miR-338 promoted OL maturation by increasing the number of MBP+ cells significantly (p<0.01). Taken together, the results demonstrate the efficacy of nanofibers in providing topographical cues and microRNA reverse transfection to direct OPC differentiation. Such scaffolds may find useful applications in directing oligodendrocyte differentiation and myelination for treatment of CNS pathological conditions that require remyelination.

MicroRNAs and Heart Failure Diagnosis

See related article, pages 1035–1039 Heart disease is the greatest noninfectious health hazard ever to confront the human race. It is the leading cause of death in industrialized nations, and, accordingly, it is responsible for a huge economic burden to society. It is estimated that 5 million Americans have heart failure (HF), a syndrome with mortality of approximately 50% at 5 years.1 Now, evidence indicates that the epidemic of HF is extending to the developing world. The road to advances in the treatment of HF is littered with failures, and many new means of diagnosing and tracking disease progression have disappointed. Therefore, there is an urgent need to identify novel mechanisms, markers, and therapeutic targets in HF pathogenesis. MicroRNAs (miRNAs or miRs) are among the most exciting areas in medicine and science today. MicroRNAs are endogenous, highly conserved, ≈22-nucleotide-long noncoding RNAs that regulate the stability and subsequent translation of nascent mRNA transcripts. Generally, miRs inhibit protein translation and/or promote mRNA degradation by base-pairing with 3′ untranslated regions within a transcript. MicroRNAs were first discovered in 1993 in Caenorhabditis elegans and are known to be critical to early vertebrate development and in an increasing number of basic molecular processes. Over the last few years, miRs have emerged as important mechanisms in human disease, including cardiovascular disease, diabetes, and cancer.2–6 Now, there is burgeoning interest in testing whether miRs could serve as markers for disease progression or prognostication. Indeed, in the cancer literature, strategies are emerging to use miR profiles for diagnosis and risk stratification. Currently, miR profiling is being evaluated in prostate cancer, head and neck squamous cell carcinomas, breast cancer, acute lymphoblastic leukemia, malignant mesothelioma, and other tumors.2,7–10 Most clinical studies to date on miRs have relied on tissue-based measurement of miR abundances, but miRs are …

Impact of clinical and histologic correlates of maternal and fetal inflammatory response on gestational age in preterm births

Objective. To evaluate the impact of clinical and histopathologic correlates related to maternal and fetal inflammatory responses (MIR and FIR) on degree of preterm birth.Methods. Pathology reports and clinical data from 577 singleton preterm births (<37 weeks of gestation) that took place between 1998 and 2004 were analyzed according to decreasing gestational age (≥33 weeks, 29–32 weeks, and <29 weeks). MIR was defined by presence of subchorionitis, chorioamnionitis, deciduitis, or free membranitis; FIR was defined by presence of funisitis or chorionic plate vasculitis. The associations between MIR alone and MIR with FIR and gestational age subgroups were assessed using logistic regression.Results. The presence of FIR in addition to MIR was more strongly associated with degree of prematurity than the presence of MIR alone, especially for those born at <29 weeks (OR = 10.1 (95% CI 4.3–23.7) and OR = 5.3 (95% CI 2.3–12.5), respectively). These associations remained significant after adjusting for maternal race, clinical signs of chorioamnionitis, medically indicated birth, and intrapartum corticosteroid, tocolysis and antibiotic use, and after stratification by clinical signs of chorioamnionitis and medically indicated birth.Conclusions. The combined presence of MIR and FIR is associated with a higher risk of extreme preterm birth (<29 weeks) than MIR alone, suggesting a contributory role of FIR in the pathophysiology of preterm birth.

Neuronal Expression of the ERM-like Protein MIR in Rat Brain and Its Localization to Human Chromosome 6

The ERM proteins, ezrin, radixin, and moesin, regulate cell motility by linking cortical F-actin to the plasma membrane in different cell types. Myosin regulatory light chain interacting protein (MIR) is a recently cloned ERM-like protein which was shown to be involved in neurite outgrowth. Here we have studied the occurrence and expression of MIR in rats during brain development. As shown using Western blotting, MIR is present in different regions both in developing and adult brain. Immunohistochemistry and double labelling studies showed that MIR is localized especially to neurons in hippocampus and cerebellum. A search using the gene bank showed that the MIR gene localised to human chromosome 6 in the interval 6p22.3–23, the loss of which is characterized by mental retardation and different malformations in man. The presence of MIR in brain neurons during development together with its known effects on neurite outgrowth suggest an important function of the protein in the regulation of nerve cell motility and cytoskeletal interactions.