miRNA Expression In Cells Research Articles

Reduced Expression of miR-146a Potentiates Intestinal Inflammation following Alcohol and Burn Injury.

MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively regulate gene expression. Within the intestinal epithelium, miRNAs play a critical role in gut homeostasis, and aberrant miRNA expression has been implicated in various disorders associated with intestinal inflammation and barrier disruption. In this study, we sought to profile changes in intestinal epithelial cell miRNA expression after alcohol and burn injury and elucidate their impact on inflammation and barrier integrity. Using a mouse model of acute ethanol intoxication and burn injury, we found that small intestinal epithelial cell expression of miR-146a is significantly decreased 1 d following injury. Using invitro studies, we show that reduced miR-146a promotes intestinal epithelial cell inflammation by promoting p38 MAPK signaling via increased levels of its target TRAF6 (TNFR-associated factor 6). Furthermore, we demonstrate that invivo miR-146a overexpression significantly inhibits intestinal inflammation 1 d following combined injury and potentially supports intestinal barrier homeostasis. Overall, this study highlights the important impact that miRNA expression can have on intestinal homeostasis and the valuable potential of harnessing aberrant miRNA expression as a therapeutic target to control intestinal inflammation.

MicroRNA Expression of Renal Proximal Tubular Epithelial Cells and Their Extracellular Vesicles in an Inflammatory Microenvironment In Vitro.

Renal proximal tubular epithelial cells (PTCs) are central players during renal inflammation. In response to inflammatory signals, PTCs not only self-express altered mRNAs, microRNAs (miRNAs), proteins, and lipids, but also release altered extracellular vesicles (EVs). These EVs also carry inflammation-specific cargo molecules and are key players in cell-cell-communication. Understanding the precise molecular and cellular mechanisms that lead to inflammation in the kidney is the most important way to identify early targets for the prevention or treatment of acute kidney injury. Therefore, highly purified human PTCs were used as an in vitro model to study the cellular response to an inflammatory microenvironment. A cytokine-induced inflammatory system was established to analyze different miRNA expression in cells and their EVs. In detail, we characterized the altered miR expression of PTCs and their released EVs during induced inflammation and showed that 12 miRNAs were significantly regulated in PTCs (6 upregulated and 6 downregulated) and 9 miRNAs in EVs (8 upregulated and 1 downregulated). We also showed that only three of the miRNAs were found to overlap between cells and EVs. As shown by the KEGG pathway analysis, these three miRNAs (miR-146a-5p, miR-147b, and miR-155-5p) are functionally involved in the regulation of the Toll-like receptor signaling pathway and significantly correlated with the inflammatory mediators IL6 and ICAM1 released by stimulated PTCs. Especially with regard to a possible clinical use of miRs as new biomarkers, an accurate characterization of the miR expression altered during inflammatory processes is of enormous importance.

Abstract 3781: NF-κB classical and alternative signaling differentially regulate miRNA expression in ovarian cancer

Abstract The NF-κB signaling pathway has been shown to contribute to epithelial ovarian cancer (EOC) through its classical and alternative pathways, characterized by the RelA and RelB transcription factors respectively. Previous studies have highlighted the role of RelA in sustaining the proliferative cancer cell population, while RelB aids in promoting the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that promote disease relapse. In further characterizing the downstream effects of NF-κB signaling on EOC, we hypothesize NF-κB signaling differentially regulates the expression of several microRNAs (miRNAs) to promote TIC survival and proliferation through its classical and alternative pathways. miRNAs comprise a subset of small, noncoding RNAs that regulate gene expression, making them amenable for therapeutic targeting. Inducible shRNA stably expressed in OV90 EOC cells to knockdown RelA or RelB were analyzed by miR-seq to identify the differential expression of miRNAs in cells grown in TIC vs adherent (adh) conditions. Several miRNAs were differentially expressed in these conditions with RelA or RelB knockdown; we identified and validated the expression of two candidate miRNAs: hsa-miR-452-5p and hsa-miR-335-5p. We observed the decreased expression of hsa-miR-452-5p when either RelA or RelB is knocked down, while also observing the increased expression of hsa-miR-335-5p when RelA is knocked down. By inhibiting miR-452-5p in conjunction with disrupting NF-κB activity, we found changes in cell viability, sphere formation, and expression of the stem cell marker ALDH. Understanding the role of miRNA signaling in the context of NF-κB will better define the transcriptional roles of RelA and RelB in EOC. Ongoing work will further characterize the downstream targets of these miRNAs as potential therapeutic targets. Citation Format: Rahul D. Kamdar, Brittney S. Harrington, Soumya Korrapati, Emma Attar, Nathan Wong, Carrie D. House, Christina M. Annunziata. NF-κB classical and alternative signaling differentially regulate miRNA expression in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3781.

Expression Microdissection for the Analysis of miRNA in a Single-Cell Type

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.

Esrrb Regulates Specific Feed-Forward Loops to Transit From Pluripotency Into Early Stages of Differentiation.

Characterization of pluripotent states, in which cells can both self-renew or differentiate, with the irreversible loss of pluripotency, are important research areas in developmental biology. Although microRNAs (miRNAs) have been shown to play a relevant role in cellular differentiation, the role of miRNAs integrated into gene regulatory networks and its dynamic changes during these early stages of embryonic stem cell (ESC) differentiation remain elusive. Here we describe the dynamic transcriptional regulatory circuitry of stem cells that incorporate protein-coding and miRNA genes based on miRNA array expression and quantitative sequencing of short transcripts upon the downregulation of the Estrogen Related Receptor Beta (Esrrb). The data reveals how Esrrb, a key stem cell transcription factor, regulates a specific stem cell miRNA expression program and integrates dynamic changes of feed-forward loops contributing to the early stages of cell differentiation upon its downregulation. Together these findings provide new insights on the architecture of the combined transcriptional post-transcriptional regulatory network in embryonic stem cells.

The emerging role of miRNAs in the pathogenesis of COVID-19: Protective effects of nutraceutical polyphenolic compounds against SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause immunosuppression and cytokine storm, leading to lung damage and death. The clinical efficacy of anti-SARS-CoV-2 drugs in preventing viral entry into host cells and suppressing viral replication remains inadequate. MicroRNAs (miRNAs) are crucial to the immune response to and pathogenesis of coronaviruses, such as SARS-CoV-2. However, the specific roles of miRNAs in the life cycle of SARS-CoV-2 remain unclear. miRNAs can participate in SARS-CoV-2 infection and pathogenesis through at least four possible mechanisms: 1. host cell miRNA expression interfering with SARS-CoV-2 cell entry, 2. SARS-CoV-2-derived RNA transcripts acting as competitive endogenous RNAs (ceRNAs) that may attenuate host cell miRNA expression, 3. host cell miRNA expression modulating SARS-CoV-2 replication, and 4. SARS-CoV-2-encoded miRNAs silencing the expression of host protein-coding genes. SARS-CoV-2-related miRNAs may be used as diagnostic or prognostic biomarkers for predicting outcomes among patients with SARS-CoV-2 infection. Furthermore, accumulating evidence suggests that dietary polyphenolic compounds may protect against SARS-CoV-2 infection by modulating host cell miRNA expression. These findings have major implications for the future diagnosis and treatment of COVID-19.

HCV Proteins Modulate the Host Cell miRNA Expression Contributing to Hepatitis C Pathogenesis and Hepatocellular Carcinoma Development.

Simple SummaryAccording to the last estimate by the World Health Organization (WHO), more than 71 million individuals have chronic hepatitis C worldwide. The persistence of HCV infection leads to chronic hepatitis, which can evolve into liver cirrhosis and ultimately into hepatocellular carcinoma (HCC). Although the pathogenic mechanisms are not fully understood, it is well established that an interplay between host cell factors, including microRNAs (miRNA), and viral components exist in all the phases of the viral infection and replication. Those interactions establish a complex equilibrium between host cells and HCV and participate in multiple mechanisms characterizing hepatitis C pathogenesis. The present review aims to describe the role of HCV structural and non-structural proteins in the modulation of cellular miRNA during HCV infection and pathogenesis.Hepatitis C virus (HCV) genome encodes for one long polyprotein that is processed by cellular and viral proteases to generate 10 polypeptides. The viral structural proteins include the core protein, and the envelope glycoproteins E1 and E2, present at the surface of HCV particles. Non-structural (NS) proteins consist of NS1, NS2, NS3, NS4A, NS4B, NS5a, and NS5b and have a variable function in HCV RNA replication and particle assembly. Recent findings evidenced the capacity of HCV virus to modulate host cell factors to create a favorable environment for replication. Indeed, increasing evidence has indicated that the presence of HCV is significantly associated with aberrant miRNA expression in host cells, and HCV structural and non-structural proteins may be responsible for these alterations. In this review, we summarize the recent findings on the role of HCV structural and non-structural proteins in the modulation of host cell miRNAs, with a focus on the molecular mechanisms responsible for the cell re-programming involved in viral replication, immune system escape, as well as the oncogenic process. In this regard, structural and non-structural proteins have been shown to modulate the expression of several onco-miRNAs or tumor suppressor miRNAs.

Analysis of microRNA expression profiles in human bronchial epithelial cells infected by Chlamydia psittaci

BackgroundChlamydia psittaci is a pathogen of birds that can cause zoonotic disease in mammals including pneumonia in humans. MicroRNAs (miRNAs) are a class of small non-coding RNA fragments with a length of about 22 nt, which play an important role in regulating gene expression after transcription. Chlamydia infection can cause changes in host cell miRNA expression, but the potential biological function of miRNAs in C. psittaci infection and pathogenesis is not well understood. MethodsSmall RNA sequencing (sRNA-Seq) technology was used to characterise miRNA expression in human bronchial epithelial (HBE) cells after C. psittaci infection, and differentially expressed miRNAs were identified. Candidate target genes for these miRNAs were then functionally annotated by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The sRNA-Seq results were partially validated by quantitative real time polymerase chain reaction (qRT-PCR) and miRNA-target networks were constructed using visualization software. ResultsWe identified 151 differentially expressed miRNAs (46 known miRNAs and 105 novel miRNAs) in C. psittaci-infected HBE cells, of which 140 were upregulated and 11 were downregulated. Of these, 17 known miRNAs were significantly upregulated and two were downregulated using P < 0.05 and |log2FoldChange|>1.5 as threshold criteria. GO enrichment results showed that the predicted targets of these differentially expressed miRNAs were mainly involved in transcriptional regulation and ATP binding. KEGG pathway analysis suggested that the candidate target genes were involved in several important signaling pathways such as MAPK, ErbB, cGMP-PKG, cAMP, mTOR, GNRH, oxytocin, PI3K-Akt and AMPK, which are primarily related to biological processes such as transcription and signal transduction. The qRT-PCR results for miR-2116–3p, miR-3195, miR-663a, miR-10401–5p, miR-124–3p, miR-184, miR-744–5p and hsa-miR-514b-5p were consistent with the sRNA-Seq data. ConclusionsA large amount of miRNA expression profile data relating to C. psittaci infection was obtained, which provides a useful experimental and theoretical basis for further understanding the pathogenic mechanisms of C. psittaci infection.

IN YOUNG WOMEN WITH POOR OVARIAN RESPONSE (POR), MICRORNA EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR (PBMN) CELLS IS ALTERED COMPARED TO NORMO-RESPONDER CONTROLS

Micro RNAs (miRNAs) are a large family of short (21 nucleotide), evolutionarily conserved, non-coding RNAs. They repress expression of their target messenger RNAs (mRNAs) by causing degradation and/or inhibition of translation. Micro RNAs play a central role in the regulation of gene expression in a number of cell types and tissues, and altered miRNA expression is implicated in the pathogenesis of numerous disorders. The aim of the current study was to determine whether peripheral somatic cell miRNA expression show unique characteristics in women with reproductive disorders associated with abnormal follicular development.

Psoralen alleviates high glucose-induced HK-2 cell injury by inhibition of Smad 2 signaling via upregulation of microRNA 874

BackgroundDiabetic nephropathy (DN) causes the vast proportion of excess mortality for patients with diabetes. Novel therapeutic approaches slowing down its incidence is still lacking. Psoralen is the major active ingredient of Psoralea corylifolia Linn. (PCL), which was used to treat a number of diseases. In this study, we aimed to investigate whether psoralen could alleviate DN using in vitro model.MethodsCell viability assay and immunofluorescence were used to evaluate the effect of psoralen on high glucose (HG)-stimulated human kidney HK-2 cells (48 h). RT-qPCR was used to detect the expressions of miRNA in cells. Cell transfection, apoptosis assay, inflammatory cytokines detection and Western blot were further performed to explore the underlying molecular mechanisms.ResultsHG-induced toxicity of HK-2 cells was alleviated by psoralen. Meanwhile, the secretion of inflammatory cytokines and extracellular matrix (ECM) accumulation induced by HG in HK-2 cells were also decreased by psoralen. In addition, the expression of miR-874 in HK-2 cells was significantly upregulated by psoralen. Western blot assays indicated that psoralen could reverse HG-induced increase of TLR-4/NF-κB and Smad2 via upregulation of miR-874.ConclusionThis study demonstrated that psoralen could significantly alleviate HG-induced HK-2 cell injury via upregulation of miR-874. In addition, HG-induced increase of TLR-4/NF-κB and Smad2 was revered by psoralen. Therefore, psoralen might serve as an agent for the treatment of DN.

MicroRNA Profiling Reveals Distinct Profiles for Tissue-Derived and Cultured Endothelial Cells

Endothelial plasticity enables the cells to switch their phenotype according to the surrounding vascular microenvironment. MicroRNAs (miRNAs) are small noncoding RNAs that control endothelial plasticity. The objective of this study was to investigate the differences in miRNA profiles of tissue-derived cells and cultured endothelial cells. To this end, miRNA expression was profiled from freshly isolated tissue-derived human vascular endothelial cells and endothelial cells cultured until cellular senescence using miRNA sequencing. In addition, the data was searched for putative novel endothelial miRNAs and miRNA isoforms. The data analysis revealed a striking change in endothelial miRNA profile as the cells adapted from tissue to cell culture environment and the overall miRNA expression decreased significantly in cultured compared to tissue-derived endothelial cells. In addition to changes in mechanosensitive miRNA expression, alterations in senescence-associated and endothelial-to-mesenchymal-transition-associated miRNAs were observed in aging cells. Collectively, the data illustrates the adaptability of endothelial cell miRNA expression that mirrors prevailing cellular environment.

MiRNAs in the alga Chlamydomonas reinhardtii are not phylogenetically conserved and play a limited role in responses to nutrient deprivation

The unicellular alga Chlamydomonas reinhardtii contains many types of small RNAs (sRNAs) but the biological role(s) of bona fide microRNAs (miRNAs) remains unclear. To address their possible function(s) in responses to nutrient availability, we examined miRNA expression in cells cultured under different trophic conditions (mixotrophic in the presence of acetate or photoautotrophic in the presence or absence of nitrogen). We also reanalyzed miRNA expression data in Chlamydomonas subject to sulfur or phosphate deprivation. Several miRNAs were differentially expressed under the various trophic conditions. However, in transcriptome analyses, the majority of their predicted targets did not show expected changes in transcript abundance, suggesting that they are not subject to miRNA-mediated RNA degradation. Mutant strains, defective in sRNAs or in ARGONAUTE3 (a key component of sRNA-mediated gene silencing), did not display major phenotypic defects when grown under multiple nutritional regimes. Additionally, Chlamydomonas miRNAs were not conserved, even in algae of the closely related Volvocaceae family, and many showed features resembling those of recently evolved, species-specific miRNAs in the genus Arabidopsis. Our results suggest that, in C. reinhardtii, miRNAs might be subject to relatively fast evolution and have only a minor, largely modulatory role in gene regulation under diverse trophic states.

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy.

MicroRNAs (miRNAs) are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS) reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.

Functional Screening Identifies Human miRNAs that Modulate Adenovirus Propagation in Prostate Cancer Cells.

Oncolytic adenoviruses represent a novel class of anticancer agents. Their efficacy in killing cancer cells is variable, suggesting that there is room for improvement. Host miRNAs have been shown to play important roles in susceptibility of cells to replication of different viruses. This study investigated if adenovirus replication in human prostate cancer cells is influenced by host cell miRNA expression. To this end, human miRNA expression in response to adenovirus infection was analyzed, and functional screens for lytic adenovirus replication were performed using synthetic miRNA mimic and inhibitor libraries. Adenovirus infection generally reduced miRNA expression. On top of this nonspecific interference with miRNA biogenesis, a set of miRNAs, including in particular miR-222, was found specifically reduced. Another set of miRNAs was found to promote adenovirus-induced death of prostate cancer cells. In most cases, this did not stimulate adenovirus propagation. The exception was miR-26b. Overexpression of miR-26b inhibited adenovirus-induced NF-κB activation, augmented adenovirus-mediated cell death, increased adenovirus progeny release, and promoted adenovirus propagation and spread in several human prostate cancer cell lines. This suggests that miR-26b is particularly useful to be combined with oncolytic adenovirus for more effective treatment of prostate cancer.

Green Tea Bioactive Polyphenol Epigallocatechin-3-O-Gallate in Osteoarthritis : Current Status and Future Perspectives

Green Tea Bioactive Polyphenol Epigallocatechin-3-O-Gallate in Osteoarthritis : Current Status and Future Perspectives

Human papillomavirus E6 and E7 oncoproteins affect the expression of cancer-related microRNAs: additional evidence in HPV-induced tumorigenesis.

Human papillomaviruses (HPVs) are the causative agents of cervical cancer and are also associated with other types of cancers. HPVs can modulate microRNAs (miRNAs) expressed by infected cells. The production of extracellular vesicles is deregulated in cancer, and their cargo delivered to the microenvironment can promote tumorigenesis. The involvement of HPV oncoproteins on miRNA expression in cells and exosomes was analyzed in keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38). MiRNAs were investigated through the TaqMan Array Human MicroRNA Cards, followed by real-time RT-PCR assay for specific miRNAs. Selected miRNA targets were analyzed by Western blot and correlated to the HPV oncoproteins by specifically silencing E6 and E7 expression. Exosomes, isolated from K16 and K38 supernatants by differential centrifugations, were quantified through the vesicle-associated acetylcholinesterase activity. MiRNAs deregulated in K16 and K38 cells were identified. HPV-16 and/or HPV-38 E6 and E7 single proteins can modify the expression of selected miRNAs involved in the tumorigenesis, in particular miR-18a, -19a, -34a and -590-5p. The analysis of the content of exosomes isolated from HPV-positive cells revealed the presence of E6 and E7 mRNAs and few miRNAs. MiR-222, a key miRNA deregulated in many cancers, was identified in exosomes from K16 cells. HPV E6 and/or E7 oncoprotein expression can induce the deregulation of some miRNAs. Through the production and function of exosomes, HPV oncogenes as well as HPV-deregulated miRNAs can potentiate the virus oncogenic effects in the tumor cell microenvironment.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3´ open reading frame than the 5´ non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia’s antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced) mechanism.

Abstract 143: Exosomal miRNA Profiling Following Ischaemic Stroke: Clinical and Pre-clinical Data

Introduction: MicroRNA (miRNA) profiles are altered in patients with stroke. Recently, active inter-cellular transport of miRNAs in extracellular vesicles (EV), such as exosomes has been shown. We assessed whether miRNAs packaged in EVs differed in patients with stroke and patients without stroke and then performed in vitro and in vivo experiments to assess potential significance of dysregulated miRNAs. Methods: We recruited patients with suspected stroke. Serum was obtained at 48h post-stroke. EVs were isolated from 200μL serum before RNA extraction. A miRNA microarray was performed (Openarray™ platform) (39 patients) to identify significantly dysregulated miRNAs in stroke vs non-stroke patients. Results were validated by Taqman™ real time quantitative polymerase chain reaction of specific miRNA (17 miRNAs, 169 patients). I n vitro studies of selected miRNAs were performed on a rat neuronal cell line (B50) and a rat glial cell line (B92) subjected to 9h hypoxia (1% O 2 ) and serum starvation with 24h reoxygenation in complete media (n=4-5/group). In vivo , total miRNA expression was quantified in serum (n=4) and in peri-infarct tissue of adult spontaneously hypertensive stroke-prone rats (SHRSP) following 45min transient middle cerebral artery occlusion (tMCAO) (n=6/group). Results: The microarray identified 26 dysregulated miRNAs in stroke patients. Using Taqman™ levels of miRNAs 17, 20b and 93 were observed to be significantly (p&lt;0.05) increased in stroke vs non-stroke patients. Patients with small vessel disease consistently had the highest levels. In vitro , a trend towards increased exosomal expression of miRNAs-17, 93 and 20b in B50 hypoxic cells was in contrast to small changes in total cell miRNA expression (vs. normoxic cells). In vivo , tMCAO did not induce significant modulation of miRNAs 17, 93 or 20b expression in SHRSP brain either 24h or 72h post-tMCAO. EVs isolated from the serum showed a significant increase in miRNA-93 expression at 72h post-tMCAO (p&lt;0.05 vs. sham animals). Conclusions: We have identified and validated changes in EV packaged miRNA expression in patients with stroke, particularly in small vessel disease. Our pre-clinical experiments corroborate the human data and support a functional role for these findings.

Molecular mechanism of anticancer effect of Sclerotium rolfsii lectin in HT29 cells involves differential expression of genes associated with multiple signaling pathways: A microarray analysis.

Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Galβ1-3GalNAc-α-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin.