MicroRNA Regulation in Extreme Environments:Differential Expression of MicroRNAs in the Intertidal Snail Littorina littorea During Extended Periods of Freezing and Anoxia

Several recent studies of vertebrate adaptation to environmental stress have suggested roles for microRNAs (miRNAs) in regulating glo- bal suppression of protein synthesis and/or restructuring protein expression patterns. The present study is the first to characterize stress-responsive alterations in the expression of miRNAs during natural freezing or anoxia exposures in an invertebrate species, the intertidal gastropod Littorina littorea. These snails are exposed to anoxia and freezing conditions as their environment constantly fluctuates on both a tidal and seasonal basis. The expression of selected miRNAs that are known to influence the cell cycle, cellular signaling pathways, carbohydrate metabolism and apoptosis was evaluated using RT-PCR. Compared to controls, significant changes in expression were observed for miR-1a-1, miR-34a and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-125b, miR-29b and miR-2a in foot muscle after freezing exposure at 6 °C for 24 h (P < 0.05). In addition, in response to anoxia stress for 24 h, significant changes in expression were also observed for miR-1a-1, miR-210 and miR-29b in hepatopancreas and for miR-1a-1, miR-34a, miR-133a, miR-29b and miR-2a in foot muscle (P < 0.05). Moreover, protein expression of Dicer, an enzyme responsible for mature microRNA processing, was increased in foot muscle during freezing and anoxia and in hepatopancreas during freezing. Alterations in expression of these miRNAs in L. littorea tissues may contribute to organismal survival under freezing and anoxia.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

Induction of Antioxidant and Heat Shock Protein Responses During Torpor in the Gray Mouse Lemur,Microcebus murinus

A natural tolerance of various environmental stresses is typically supported by various cytoprotective mechanisms that protect macromolecules and promote extended viability. Among these are antioxidant defenses that help to limit damage from reactive oxygen species and chaperones that help to minimize protein misfolding or unfolding under stress conditions. To understand the molecular mechanisms that act to protect cells during primate torpor, the present study characterizes antioxidant and heat shock protein(HSP) responses in various organs of control(aroused)and torpid gray mouse lemurs, Microcebus murinus. Protein expression of HSP70 and HSP90 a was elevated to 1.26 and 1.49 fold, respectively, in brown adipose tissue during torpor as compared with control animals, whereas HSP60 in liver of torpid animals was 1.15 fold of that in control(P 0.05). Among antioxidant enzymes, protein levels of thioredoxin 1 were elevated to 2.19 fold in white adipose tissue during torpor, whereas Cu–Zn superoxide dismutase 1 levels rose to 1.1 fold in skeletal muscle(P 0.05). Additionally, total antioxidant capacity was increased to 1.6 fold in liver during torpor(P 0.05), while remaining unchanged in the five other tissues. Overall, our data suggest that antioxidant and HSP responses are modified in a tissue-specific manner during daily torpor in gray mouse lemurs. Furthermore, our data also show that cytoprotective strategies employed during primate torpor are distinct from the strategies in rodent hibernation as reported in previous studies.     

Global Protein Conjugation by Ubiquitin-Like-Modifiers during Ischemic Stress Is Regulated by MicroRNAs and Confers Robust Tolerance to Ischemia

Hibernation torpor provides an excellent model of natural tolerance to ischemia. We have previously shown that massive global SUMOylation occurs during hibernation torpor in ground squirrels. We have also shown that overexpression of Ubc9, SUMO-1, or SUMO-2/3 provides protection against ischemic damage in cell lines and cortical neurons exposed to oxygen/glucose deprivation, and in mice exposed to middle cerebral artery occlusion. We have now extended our study to other Ubiquitin-Like- Modifiers (ULMs), which have multiple cellular functions during stress, in order to assess the possibility that they also have roles in tolerance to ischemia. We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor. By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals. In addition, we showed that these miRNAs are involved in the expression of various ULM proteins and their global conjugation to proteins. We observed that inhibition of the miR-200 family and/or miR-182 family miRNA activities in SHSY5Y cells increases global protein conjugation by the above ULMs and makes these cells more tolerant to OGD-induced cell death. This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.     

MicroRNA-494, Upregulated by Tumor Necrosis Factor-α, Desensitizes Insulin Effect in C2C12 Muscle Cells

Chronic inflammation is fundamental for the induction of insulin resistance in the muscle tissue of vertebrates. Although several miRNAs are thought to be involved in the development of insulin resistance, the role of miRNAs in the association between inflammation and insulin resistance in muscle tissue is poorly understood. Herein, we investigated the aberrant expression of miRNAs by conducting miRNA microarray analysis of TNF-α-treated mouse C₂C₁₂ myotubes. We identified two miRNAs that were upregulated and six that were downregulated by a >1.5-fold change compared to normal cells. Among the findings, qRT-PCR analysis confirmed that miR-494 is consistently upregulated by TNF-α-induced inflammation. Overexpression of miR-494 in CHO_IR/IRS1 and C₂C₁₂ myoblasts suppressed insulin action by down-regulating phosphorylations of GSK-3α/β, AS160 and p70S6K, downstream of Akt. Moreover, overexpression of miR-494 did not regulate TNF-α-mediated inflammation . Among genes bearing the seed site for miR-494, RT-PCR analysis showed that the expression of Stxbp5, an inhibitor of glucose transport, was downregulated following miR-494 inhibition. In contrast, the expression of PTEN decreased in the cells analyzed, thus showing that both positive and negative regulators of insulin action may be simultaneously controlled by miR-494. To investigate the overall effect of miR-494 on insulin signaling, we performed a PCR array analysis containing 84 genes related to the insulin signaling pathway, and we observed that 25% of genes were downregulated (P<0.05) and 11% were upregulated (P<0.05). These results confirm that miR-494 might contribute to insulin sensitivity by positive and negative regulation of the expression of diverse genes. Of note, PCR array data showed downregulation of Slc2A4, a coding gene for Glut4. Altogether, the present study concludes that the upregulation of miR-494 expression by TNF-α-mediated inflammation exacerbates insulin resistance. Therefore, we suggest that miR-494 could prove an important target for the diagnosis and therapy of inflammation-mediated insulin resistance in muscle.     

MicroRNAs Regulate Human Adipocyte Lipolysis: Effects of miR-145 Are Linked to TNF-α

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.     

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3′ UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.     

MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial–mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA–target interactions. Knockdown of miR-382, which was up-regulated by TGFβ1, attenuated TGFβ1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3′-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFβ1.     

The impact of cold acclimation and hibernation on antioxidant defenses in the ground squirrel (Spermophilus citellus): An update

Any alteration in oxidative metabolism is coupled with a corresponding response by an antioxidant defense (AD) in appropriate subcellular compartments. Seasonal hibernators pass through circannual metabolic adaptations that allow them to either maintain euthermy (cold acclimation) or enter winter torpor with body temperature falling to low values. The present study aimed to investigate the corresponding pattern of AD enzyme protein expressions associated with these strategies in the main tissues involved in whole animal energy homeostasis: brown and white adipose tissues (BAT and WAT, respectively), liver, and skeletal muscle. European ground squirrels (Spermophilus citellus) were exposed to low temperature (4 ± 1 °C) and then divided into two groups: (1) animals fell into torpor (hibernating group) and (2) animals stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). We examined the effects of cold acclimation and hibernation on the tissue-dependent protein expression of four enzymes which catalyze the two-step detoxification of superoxide to water: superoxide dismutase 1 and 2 (SOD 1 and 2), catalase (CAT), and glutathione peroxidase (GSH-Px). The results showed that hibernation induced an increase of AD enzyme protein expressions in BAT and skeletal muscle. However, AD enzyme contents in liver were largely unaffected during torpor. Under these conditions, different WAT depots responded by elevating the amounts of specific enzymes, as follows: SOD 1 in retroperitoneal WAT, GSH-Px in gonadal WAT, and CAT in subcutaneous WAT. Similar perturbations of AD enzymes contents were seen in all tissues during cold acclimation, often in a time-dependent manner. It can be concluded that BAT and muscle AD capacity undergo the most dramatic changes during both cold acclimation and hibernation, while liver is relatively unaffected by either condition. Additionally, this study provides a basis for further metabolic study that will illuminate the causes of these tissue-specific AD responses, particularly the novel finding of distinct responses by different WAT depots in hibernators.     

microRNA profiling in Epstein-Barr virus-associated B-cell lymphoma

The Epstein–Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19–26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes.     

Regulatory roles of miR-155 and let-7b on the expression of inflammation-related genes in THP-1 cells: effects of fatty acids

The main aim of this investigation was to study the regulatory roles of let-7b and miR-155-3p on the expression of inflammation-associated genes in monocytes, macrophages, and lipopolysaccharide (LPS)-activated macrophages (AcM). A second goal was to analyze the potential modulatory roles of different fatty acids, including oleic, palmitic, eicosapentaenoic (EPA), and docosahexaenoic (DHA), on the expression of these miRNAs in the three cell types. This hypothesis was tested in human acute monocytic leukemia cells (THP-1), which were differentiated into macrophages with 2-O-tetradecanoylphorbol-13-acetate (TPA) and further activated with LPS for 24 h. Monocytes, macrophages, and AcM were transfected with a negative control, or mimics for miR-155-3p and miR-let-7b-5p. The expression of both miRNAs and some proinflammatory genes was analyzed by qRT-PCR. Interestingly, let-7b mimic reduced the expression of IL6 and TNF in monocytes, and SERPINE1 expression in LPS-activated macrophages. However, IL6, TNF, and SERPINE1 were upregulated in macrophages by let-7b mimic. IL6 expression was higher in the three types of cells after transfecting with miR-155-3p mimic. Similarly, expression of SERPINE1 was increased by miR-155-3p mimic in monocytes and macrophages. However, TLR4 was downregulated by miR-155-3p in monocytes and macrophages. Regarding the effects of the different fatty acids, oleic acid increased the expression of let-7b in macrophages and AcM and also increased the expression of miR-155 in monocytes when compared with DHA but not when compared with non-treated cells. Overall, these results suggest anti- and proinflammatory roles of let-7b and miR-155-3p in THP-1 cells, respectively, although these outcomes are strongly dependent on the cell type. Noteworthy, oleic acid might exert beneficial anti-inflammatory effects in immune cells (i.e., non-activated and LPS-activated macrophages) by upregulating the expression of let-7b.     

miR-135b Contributes to the Radioresistance by Targeting GSK3β in Human Glioblastoma Multiforme Cells

Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). Recent data strongly suggests the important role of miRNAs in cancer progression and therapeutic response. Here, we have established a radioresistant human GBM cell line U87R derived from parental U87 and found miR-135b expression was upregulated in U87R cells. miR-135b knockdown reversed radioresistance of U87R cells, and miR-135b overexpression enhanced radioresistance of U87 cells. Mechanically, bioinformatics analysis combined with experimental analysis demonstrated GSK3β (Glycogen synthase kinase 3 beta) was a novel direct target of miR-135b. Moreover, GSK3β protein expression was downregulated in U87R cells and restored expression of GSK3β increased radiosensitivity of U87R cells. In addition, clinical data indicated that the expression of miR-135b or GSK3β was significantly association with IR resistance of GBM samples. Our findings suggest miR-135b is involved in the radioresistance of human GBM cells and miR-135b-GSK3β axis may be a novel candidate for developing rational therapeutic strategies for human GBM treatment.     

Long non-coding RNA TUG1/microRNA-187-3p/TESC axis modulates progression of pituitary adenoma via regulating the NF-κB signaling pathway

The molecule mechanisms of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in human diseases have been broadly studied recently, therefore, our research aimed to assess the effect of lncRNA taurine upregulated gene 1 (TUG1)/miR-187-3p/tescalcin (TESC) axis in pituitary adenoma (PA) by regulating the nuclear factor-kappa B (NF-κB) signaling pathway. We observed that TUG1 was upregulated in PA tissues and was associated with invasion, knosp grade and tumor size. TUG1 particularly bound to miR-187-3p. TUG1 knockdown inhibited cell proliferation, invasion, migration, and epithelial–mesenchymal transition, promoted apoptosis, and regulated the expression of NF-κB p65 and inhibitor of κB (IκB)-α in PA cells lines in vitro, and also inhibited tumor growth in vivo, and these effects were reversed by miR-187-3p reduction. Similarly, miR-187-3p elevation inhibited PA cell malignant behaviors and modulated the expression of NF-κB p65 and IκB-α in PA cells, and reduced in vivo tumor growth as well. TUG1 inhibition downregulated TESC, which was targeted by miR-187-3p. In conclusion, this study suggests that TUG1 sponges miR-187-3p to affect PA development by elevating TESC and regulating the NF-κB signaling pathway.Subject terms: Cell biology, Diseases     

Markers of tissue remodeling and inflammation in the white and brown adipose tissues of a model hibernator

The thirteen-lined ground squirrel is a model fat-storing hibernator that nearly doubles its weight in the fall to fuel metabolism with triglycerides throughout the winter months. Hibernator brown and white adipose tissue (BAT, WAT) are important to study in terms of their inflammatory profile and tissue remodeling mechanisms since controlled and natural regulation of these processes could inform new pharmacological interventions that limit oxidative stress and inflammation in the adipose tissues of humans suffering from obesity, promote non-shivering thermogenesis-mediated weight loss, or prevent tissue damage in transplantable organs emerging from cold-storage. Thus, markers of inflammation like cytokines and soluble receptors and tissue remodeling proteins such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were investigated in normothermic, torpid, and arousing ground squirrels. Multiplex protein assays and western blotting revealed fewer changes in WAT compared to BAT. Pro-inflammatory IL-1α levels increased during torpor and soluble epidermal growth factor receptor protein levels increased during arousal in BAT. Given their known roles in other model systems, these proteins could regulate processes like adipogenesis, lipid catabolism, or cell motility. Decreased TIMP2 levels combined with maintained MMP2 or MMP3 protein levels suggested that BAT may avoid tissue remodeling until arousal. No changes in WAT inflammatory cytokines or soluble receptors as well as decreased MMP2 levels during torpor and arousal suggested inflammation and modification to the extracellular matrix is likely suppressed in WAT. This study emphasizes the fat-but-fit nature of the hibernating ground squirrel and the ability of its fat stores to suppress inflammation.