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Pentameric ligand-gated ion channels (pLGICs) activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are expressed widely in both vertebrate and invertebrate species. One of the best characterised insect GABA-gated chloride channels is RDL, an abbreviation of 'resistance to dieldrin', that was originally identified by genetic screening in Drosophila melanogaster. Here we have cloned the analogous gene from the bumblebee Bombus terrestris audax (BtRDL) and examined its pharmacological properties by functional expression in Xenopus oocytes. Somewhat unexpectedly, the sensitivity of BtRDL to GABA, as measured by its apparent affinity (EC50), was influenced by heterologous expression conditions. This phenomenon was observed in response to alterations in the amount of cRNA injected; the length of time that oocytes were incubated before functional analysis; and by the presence or absence of a 3' untranslated region. In contrast, similar changes in expression conditions were not associated with changes in apparent affinity with RDL cloned from D. melanogaster (DmRDL). Changes in apparent affinity with BtRDL were also observed following co-expression of a chaperone protein (NACHO). Similar changes in apparent affinity were observed with an allosteric agonist (propofol) and a non-competitive antagonist (picrotoxinin), indicating that expression-depended changes are not restricted to the orthosteric agonist binding site. Interestingly, instances of expression-dependent changes in apparent affinity have been reported previously for vertebrate glycine receptors, which are also members of the pLGIC super-family. Our observations with BtRDL are consistent with previous data obtained with vertebrate glycine receptors and indicates that agonist and antagonist apparent affinity can be influenced by the level of functional expression in a variety of pLGICs., Competing Interests: Syngenta provided support in the form of salary for JG. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Understanding mechanisms of resistance to abiraterone, one of the primary drugs approved for the treatment of castration resistant prostate cancer, remains a priority. The organic anion polypeptide 1B3 (OATP1B3, encoded by SLCO1B3) transporter has been shown to transport androgens into prostate cancer cells. In this study we observed and investigated the mechanism of induction of SLCO1B3 by abiraterone. Prostate cancer cells (22Rv1, LNCaP, and VCAP) were treated with anti-androgens and assessed for SLCO1B3 expression by qPCR analysis. Abiraterone treatment increased SLCO1B3 expression in 22Rv1 cells in vitro and in the 22Rv1 xenograft model in vivo. MicroRNA profiling of abiraterone-treated 22Rv1 cells was performed using a NanoString nCounter miRNA panel followed by miRNA target prediction. TargetScan and miRanda prediction tools identified hsa-miR-579-3p as binding to the 3'-untranslated region (3'UTR) of the SLCO1B3. Using dual luciferase reporter assays, we verified that hsa-miR-579-3p indeed binds to the SLCO1B3 3'UTR and significantly inhibited SLCO1B3 reporter activity. Treatment with abiraterone significantly downregulated hsa-miR-579-3p, indicating its potential role in upregulating SLCO1B3 expression. In this study, we demonstrated a novel miRNA-mediated mechanism of abiraterone-induced SLCO1B3 expression, a transporter that is also responsible for driving androgen deprivation therapy resistance. Understanding mechanisms of abiraterone resistance mediated via differential miRNA expression will assist in the identification of potential miRNA biomarkers of treatment resistance and the development of future therapeutics.

Increased glucagon is a hallmark of diabetes and leads to worsening of the hyperglycemia, but the molecular mechanisms causing it are still unknown. We therefore investigated the possibility that microRNAs might be involved in the regulation of glucagon. Indeed, analysis of the glucagon 3' untranslated region (UTR) revealed potential binding sites for miR-320a, and using luciferase reporter assays we found that miR-320a directly targets the 3' UTRs of human and rodent glucagon. In addition, endogenous glucagon mRNA and protein expression as well as glucagon secretion were reduced in response to miR-320a overexpression, whereas inhibition of miR-320a upregulated glucagon expression. Interestingly, miR-320a expression was decreased by high glucose, and this was associated with an increase in glucagon expression in human islets and mouse αTC1-6 cells. Moreover, miR-320a overexpression completely blunted these effects. Importantly, miR-320a was also significantly downregulated in human islets of subjects with type 2 diabetes and this was accompanied by increased glucagon expression. Thus, our data suggest that glucose-induced downregulation of miR-320a may contribute to the paradoxical increase in glucagon observed in type 2 diabetes and reveal for the first time that glucagon expression is under the control by a microRNA providing novel insight into the abnormal regulation of glucagon in diabetes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ginsenoside Rb1 (GRb1), a major ingredient of ginseng, has been found to be a potential protective agent in spinal cord injury (SCI) and in activated microglia-induced neuronal injury. This study discovered that GRb1 could facilitate miR-130b-5p expression in SCI rats and Toll-like receptor 4 (TLR4; a crucial player in inflammation) was a potential target of miR-130b-5p. Hence, we further investigated whether GRb1 could relieve SCI by reducing microglia-mediated inflammatory responses and neuronal injury via miR-130b-5p/TLR4 pathways. The results showed that GRb1 alleviated SCI through inhibiting neuronal apoptosis and proinflammatory factor expression via increasing miR-130b-5p.GRb1 weakened the damage of activated microglia to neurons through upregulating miR-130b-5p. miR-130b-5p attenuated activated microglia-induced neuron injury via targeting TLR4. GRb1 inactivated TLR4/nuclear factor-κB (NF-κB) activation and inhibited proinflammatory cytokine secretion by increasing miR-130b-5p in activated microglia. As a conclusion, GRb1 alleviated SCI through reducing activated microglia-induced neuronal injury via miR-130b-5p/TLR4/NF-κB axis, providing a deep insight into the molecular basis of GRb1 in the treatment of SCI., (© 2020 Wiley Periodicals LLC.)

Since the identification of microRNAs (miRNAs) involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, miRNAs known to down-regulate the expression of the CFTR and associated proteins have been investigated as potential therapeutic targets. Here we show that miR-101-3p, targeting the 3'-UTR sequence of the CFTR mRNA, can be selectively inhibited by a peptide nucleic acid (PNA) carrying a full complementary sequence. With respect to clinical relevance of microRNA targeting, it is expected that reduction in concentration of miRNAs (the anti-miRNA approach) could be associated with increasing amounts of target mRNAs. Consistently to this hypothesis, we report that PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. Next Generation Sequencing (NGS) was performed in order to verify the effects of the anti-miR-101-3p PNA on the Calu-3 miRNome. Upon inhibition of miR-101-3p we observed a fold change (FC) expression <2 of the majority of miRNAs (403/479, 84.13%), whereas we identified a list of dysregulated miRNAs, suggesting that specific miRNA inhibition (in our case miR-101-3p) might be accompanied by alteration of expression of other miRNAs, some of them known to be involved in Cystic Fibrosis (CF), such as miR-155-5p and miR-125b-5p., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Masson SAS.)

It has been reported that butyrate played an protect role in diabetic kidney disease (DKD) while the mechanism was still not clear. Transforming growth factor-β1 (TGF-β1) is the initial factor which triggers the profibrotic signaling cascades. P311 is an RNA-binding protein, which could stimulate TGF-β1 translation in several cell types. In our study, we found that supplementary of butyrate alleviated fibrosis and suppressed the expression of TGF-β1 and P311 in the kidney of db/db mice as well as high glucose (HG)-induced SV40-MES-13 cells. Overexpression of P311 offset the inhibition of butyrate on TGF-β1 in SV40-MES-13 cells. To make clear the mechanism of butyrate in regulating P311, microRNAs (miRNAs) of the SV40-MES-13 cells were sequenced. We found that miR-7a-5p was significantly decreased in the HG-induced SV40-MES-13 cells and the kidney of db/db mice, while giving butyrate reversed this change. Besides, miR-7a-5p could specifically target the 3' UTR of P311's mRNA and suppressed the expression of P311 in the SV40-MES-13 cells. Giving miR-7a-5p inhibitor blocked the inhibition of butyrate on P311 and TGF-β1. Introducing the miR-7a-5p agomir into db/db mice alleviated renal fibrosis and inhibit the expression of P311 and TGF-β1. In conclusion, butyrate alleviated DKD by mediating the miR-7a-5p/P311/TGF-β1 pathway., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3'-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.

Guanine-rich sequences in the genomes of herpesviruses can fold into G-quadruplexes. Compared with the widely-studied G₃-quadruplexes, the dynamic G₂-quadruplexes are more sensitive to the cell microenvironment, but they attract less attention. Pseudorabies virus (PRV) is the model species for the study of the latency and reactivation of herpesvirus in the nervous system. A total of 1722 G₂-PQSs and 205 G₃-PQSs without overlap were identified in the PRV genome. Twelve G₂-PQSs from the CDS region exhibited high conservation in the genomes of the Varicellovirus genus. Eleven G₂-PQSs were 100% conserved in the repeated region of the annotated PRV genomes. There were 212 non-redundant G₂-PQSs in the 3' UTR and 19 non-redundant G₂-PQSs in the 5' UTR, which would mediate gene expression in the post-transcription and translation processes. The majority of examined G₂-PQSs formed parallel structures and exhibited different sensitivities to cations and small molecules in vitro. Two G₂-PQSs, respectively, from 3' UTR of UL5 (encoding helicase motif) and UL9 (encoding sequence-specific ori-binding protein) exhibited diverse regulatory activities with/without specific ligands in vivo. The G-quadruplex ligand, NMM, exhibited a potential for reducing the virulence of the PRV Ea strain. The systematic analysis of the distribution of G₂-PQSs in the PRV genomes could guide further studies of the G-quadruplexes' functions in the life cycle of herpesviruses.

Thyroid cancer is rapidly increasing in incidence worldwide. Although most thyroid cancer can be cured with surgery, radioactive iodine, and/or chemotherapy, thyroid cancers still recur and may become chemoresistant. Autophagy is a complex self-degradative process that plays a dual role in cancer development and progression. In this study, we found that miR-125b was downregulated in tissue samples of thyroid cancer as well as in thyroid cancer cell lines, and the expression of Foxp3 was upregulated. Further, we demonstrated that miR-125b could directly act on Foxp3 by binding to its 3' UTR and inhibit the expression of Foxp3. A negative relationship between miR-125b and Foxp3 was thus revealed. Overexpression of miR-125b markedly sensitized thyroid cancer cells to cisplatin treatment by inducing autophagy through an Atg7 pathway in vitro and in vivo. Taken together, our findings demonstrate a novel mechanism by which miR-125b has the potential to negatively regulate Foxp3 to promote autophagy and enhance the efficacy of cisplatin in thyroid cancer. miR-125 may be of therapeutic significance in thyroid cancer., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The epigenetic effects on expression of non-coding RNAs (e.g. microRNAs) of environmental toxin bisphenol A (BPA) have extended our understanding of the etiology of human reproductive disorders including hypospermatogenesis and androgen deficiency. BPA-induced miR-146a-5p is a potent regulator of endocrine and immune homeostasis, but its role in testis remain unexplored. We show here that in murine testis, miR-146a-5p was exclusively expressed in interstitial Leydig cells (LCs). This expression was significantly induced by BPA exposure. Consequently, the elevated miR-146a-5p exacerbated the deleterious effects of BPA on testicular steroidogenesis. Mechanistically, miR-146a-5p repressed the expression of Mta3, a pivotal chromatin remodeling transcription factor recently involved in controlling the steroidogenic activity, via directly targeting its 3'UTR. This repression thereafter rendered LCs more sensitive to BPA-elicited inhibitory effects. Conversely, ectopic expression of hMTA3 successfully rescued miR-146a-5p-elicited inhibitory effects on testicular steroidogenesis in BPA-challenged LCs. Taken together, the available data provide novel evidence that deregulation of testicular miR-146a-5p/Mta3 cascade mediates, at least in part, the steroidogenic dysfunction caused by BPA exposure., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glucagon-like peptide 1 receptor (GLP1R) agonists are widely used to treat diabetes. However, their function is dependent on adequate GLP1R expression, which is downregulated in diabetes. GLP1R is highly expressed on pancreatic β-cells, and activation by endogenous incretin or GLP1R agonists increases cAMP generation, which stimulates glucose-induced β-cell insulin secretion and helps maintain glucose homeostasis. We now have discovered that the highly β-cell-enriched microRNA, miR-204, directly targets the 3' UTR of GLP1R and thereby downregulates its expression in the β-cell-derived rat INS-1 cell line and primary mouse and human islets. Furthermore, in vivo deletion of miR-204 promoted islet GLP1R expression and enhanced responsiveness to GLP1R agonists, resulting in improved glucose tolerance, cAMP production, and insulin secretion as well as protection against diabetes. Since we recently identified thioredoxin-interacting protein (TXNIP) as an upstream regulator of miR-204, we also assessed whether in vivo deletion of TXNIP could mimic that of miR-204. Indeed, it also enhanced islet GLP1R expression and GLP1R agonist-induced insulin secretion and glucose tolerance. Thus, the present studies show for the first time that GLP1R is under the control of a microRNA, miR-204, and uncover a previously unappreciated link between TXNIP and incretin action., (© 2017 by the American Diabetes Association.)

Resistin is an adipocyte-derived cytokine and was named for its role in the development of insulin resistance. Increased serum resistin levels are also associated with steatohepatitis and non-alcoholic fatty liver disease. In a previous study, resistin was observed to reduce mitochondrial content and upregulate miR-34a significantly in the liver. In this study, male C57BL/6 mice were injected with agomir-34a or control agomir, and HepG2 cells were transfected with miR-34a mimics or inhibitors to assess their role in resistin-induced fat deposition. The overexpression of miR-34a increased liver and HepG2 cell TAG content, decreased mitochondrial content, changed mitochondrial morphology and impaired mitochondrial function. In contrast, a miR-34a inhibitor significantly restored the TAG content and mitochondrial transmembrane potential. A study of transcriptional regulation revealed that C/EBPβ is essential for upregulating miR-34a by resistin. Furthermore, miR-34a inhibited the PPARα signaling pathway by binding to sites in the 3'UTR of AdipoR2 genes and the AMPK pathway. Consequently, this increased the fat content and decreased the mitochondrial content in HepG2 cells. This paper reveals a novel mechanism for mitochondrial regulation, which suggests that normal mitochondrial content and function is crucial for lipid metabolism in the liver., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Puerarin has attracted increasing attention because of its beneficial effects on anti‑osteoporosis, but the molecular mechanisms underlying its actions on osteoblasts are not fully understood. The current study aimed to investigate the effect of puerarin on the cell viability and differentiation of mouse MC3T3‑E1 osteoblast‑like cells in vitro and its underlying mechanisms. The results indicated that 0.01, 0.1 and 1 mg/ml puerarin significantly promoted the viability of osteoblasts, enhanced alkaline phosphatase (ALP) activity and increased the expression of transforming growth factor‑β1, Smad2, Smad3 and Runt‑related transcription factor (Runx)2. Micro (mi)RNA target prediction programs predicted that miR‑204 may directly target Runx2. Following treatment with 0.1 mg/ml puerarin for 48 h, the expression level of miR‑204 was downregulated. Besides, miR‑204 dramatically repressed the luciferase activity of wildtype Runx2 3'‑UTR transfected cells, but not that of the mutant ones. Overexpression of miR‑204 in osteoblasts significantly decreased the protein expression of Runx2, while inhibition of miR‑204 enhanced Runx2's expression. In addition, overexpression of miR‑204 inhibited the cell viability and ALP activity of osteoblasts, while inhibition of miR‑204 had the opposite effect. The results suggested that puerarin may promote MC3T3‑E1 osteoblast‑like cell viability and differentiation, which may be related to the downregulation of miR‑204 and the following activation of Runx2.

Recent studies suggest that cytokines and microRNAs play a key role in the destruction of cartilage matrix in osteoarthritis (OA) tissues. In the current study, we focused on miR-204, which has never been explored in OA. We found that the level of miR-204 was markedly reduced in the OA cartilage tissues compared with that of normal control. Real time PCR analysis demonstrated that the level of miR-204 was markedly decreased after IL-1β treatment for 3, 6, 12 h in the normal chondrocytes and OA chondrocytes, respectively. Furthermore, overexpression of miR-204 markedly suppressed the protein levels of IL-1β, COX-2 and IL6 in human OA chondrocytes and chondrogenic SW1353 cells. Dual luciferase reporter assay demonstrated that miR-204 significantly suppressed the relative luciferase activity of pmirGLO-IL-1β-3'UTR, indicating that IL-1β was a target gene of miR-204. More importantly, treatment with IL-1β significantly enhanced the protein levels of IL-1β, COX-2 and IL6. However, overexpression of miR-204 could partially abolish such effects. In conclusion, our data demonstrate that reduced miR-204 expression enhances the destruction of the cartilage tissues among OA patients mainly through targeting IL-1β.

Metal regulation is essential for terrestrial gastropods to survive. In helicid snails, two metal-selective metallothionein (MT) isoforms with different functions are expressed. A cadmium-selective isoform (CdMT) plays a major role in Cd 2+ detoxification and stress response, whereas a copper-selective MT (CuMT) is involved in Cu homeostasis and hemocyanin synthesis. A third, non-metal-selective isoform, called Cd/CuMT, was first characterized in Cantareus aspersus. The aim of this study was to quantify the transcriptional activity of all three MT genes in unexposed and metal-exposed (Cd, Cu) embryonic Roman snails. In addition, the complete Cd/CuMT mRNA of the Roman snail (Helix pomatia) was characterized, and its expression quantified in unexposed and Cd-treated adult individuals. In embryos of Helix pomatia, the Cd/CuMT gene was induced upon Cu exposure. Its transcription levels were many times higher than that of the other two MT genes, and also exceeded by far the Cd/CuMT mRNA concentrations of adult snails. In the hepatopancreas of adult Roman snails, no Cd/CuMT could be detected at the protein level, irrespective of whether the snails had been exposed to Cd or not. This contrasts with the situation in the near relative, Cantareus aspersus. It appeared that the 3'-UTR of the Cd/CuMT mRNA differed largely between Cantareus aspersus and Helix pomatia, being larger in the latter species, with a number of putative binding sites for proteins and miRNAs known to inhibit mRNA translation. We suggest this as a possible mechanism responsible for the lack of Cd/CuMT protein expression in adult Roman snails., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

NK3 homeobox 1 (Nkx3.1), a transcription factor expressed in the prostate epithelium, is crucial for maintaining prostate cell fate and suppressing tumor initiation. Nkx3.1 is ubiquitously expressed in luminal cells of hormonally intact prostate but, upon androgen deprivation, exclusively labels a type of luminal stem cells named castration-resistant Nkx3.1-expressing cells (CARNs). During prostate cancer initiation, Nkx3.1 expression is frequently lost in both humans and mouse models. Therefore, investigating how Nkx3.1 expression is regulated in vivo is important for understanding the mechanisms of prostate stem cell specification and cancer initiation. Here, using a transgenic mouse line with destabilized GFP, we identified an 11-kb genomic region 3' of the Nkx3.1 transcription start site to be responsible for alterations in Nkx3.1 expression patterns under various physiological conditions. We found that androgen cell-autonomously activates Nkx3.1 expression through androgen receptor (AR) binding to the 11-kb region in both normal luminal cells and CARNs and discovered new androgen response elements in the Nkx3.1 3' UTR. In contrast, we found that, in Pten -/- prostate tumors, loss of Nkx3.1 expression is mediated at the transcriptional level through the 11-kb region despite functional AR in the nucleus. Importantly, the GFP reporter specifically labeled CARNs in the regressed prostate only in the presence of cell-autonomous AR, supporting a facultative model for CARN specification., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Tamoxifen-resistant (TAMR) estrogen receptor-positive (ER+) breast cancer is characterized by elevated Erb-B2 receptor tyrosine kinase 2 (ERBB2) expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the TAMR cells remain poorly understood. Herein, we reported that the ERBB2 expression is regulated at the post-transcriptional level by miR26a/b and the RNA-binding protein human antigen R (HuR), both of which associate with the 3'-UTR of the ERBB2 transcripts. We demonstrated that miR26a/b inhibits the translation of ERBB2 mRNA, whereas HuR enhances the stability of the ERBB2 mRNA. In TAMR ER+ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER+ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER+ breast cancer cells is attributable to an increase in the HuR mRNA isoform with shortened 3'-UTR, which exhibits increased translational activity. This shortening of the HuR mRNA 3'-UTR via alternative polyadenylation (APA) was observed to be dependent on cleavage stimulation factor subunit 2 (CSTF2/CstF-64), which is up-regulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR post-transcriptionally up-regulates ERBB2 expression in TAMR ER+ breast cancer cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human lif mRNA revealed several conserved sequences with conventional cis -acting elements. A 216 nucleotide containing proximal cis -element with two AUUUA pentamers and four poly-rC sequences demonstrated significant mRNA destabilizing potential, which, on treatment with PMA, showed stabilizing activity. Affinity chromatography followed by western blot and RNA co-immunoprecipitation of PMA-treated U937 extract identified Nucleolin and PCBP1 as two protein trans -factors interacting with lif mRNA, specifically to the proximal non-conventional AU-rich region. PMA induced nucleo-cytoplasmic translocation of both Nucleolin and PCBP1. RNA-dependent in vivo co-association of both these proteins with lif mRNA was demonstrated by decreased co-precipitation in the presence of RNase. Ectopic overexpression of Nucleolin showed stabilization of both intrinsic lif mRNA and gfp reporter, whereas knockdown of Nucleolin and PCBP1 demonstrated a significant decrease in both lif mRNA and protein levels. Collectively, this report establishes the stabilization of lif mRNA by PMA, mediated by the interactions of two RNA-binding proteins, Nucleolin and PCBP1 with a proximal cis -element., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The RNA-binding protein Hu antigen R (HuR) binds to AU-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs. The HuR-ARE interactions stabilize many oncogenic mRNAs that play important roles in tumorigenesis. Thus, small molecules that interfere with the HuR-ARE interaction could potentially inhibit cancer cell growth and progression. Using a fluorescence polarization (FP) competition assay, we identified the compound azaphilone-9 (AZA-9) derived from the fungal natural product asperbenzaldehyde, binds to HuR and inhibits HuR-ARE interaction (IC50 ~1.2 μM). Results from surface plasmon resonance (SPR) verified the direct binding of AZA-9 to HuR. NMR methods mapped the RNA-binding interface of HuR and identified the involvement of critical RNA-binding residues in binding of AZA-9. Computational docking was then used to propose a likely binding site for AZA-9 in the RNA-binding cleft of HuR. Our results show that AZA-9 blocks key RNA-binding residues of HuR and disrupts HuR-RNA interactions in vitro. This knowledge is needed in developing more potent AZA-9 derivatives that could lead to new cancer therapy.

MicroRNAs (miRs) are reported to play key roles in various disease models. In this study, the functional role of miR-300 in the regulation of lung injury was explored to assess the feasibility of serum miR-300 as a potential biomarker for lung injury. Firstly, the expression of miR-300 was studied in the serum of 50 lung injury patients and 50 healthy controls. And the expression of miR-300 was also explored in the serum and lung tissues of mouse models. To further explore the possible mechanism in which miR-300 may contribute to lung injury, the target genes of miR-300 were predicted by TargetScan and validated using dual luciferase reporter assay. Moreover, the expression of inflammation factors was studied after transfection of miR-300 mimics and inhibitors into A549 cells. Here, we first identified that the level of miR-300 was significantly upregulated in the blood samples of acute lung injury patients compared with healthy control. Meanwhile, miR-300 was also found to be enhanced in the blood samples and lung tissues of LPS-induced mouse models. Further study showed that miR-300 significantly suppressed the expression of IκBα and luciferase reporter assay showed that IκBα was a target gene of miR-300. More importantly, the levels of inflammatory factors, such as TNFα, COX-2, iNOS, IL-6 and IL8, were significantly upregulated accompanied by overexpression of miR-300 in A549 cells. In summary, enhanced miR-300 expression in the peripheral blood contributed to the lung injury mainly by inhibiting the expression of IκBα.

The marine dinoflagellate Cochlodinium polykrikoides is one of the most common ichthyotoxic species that causes harmful algal blooms (HABs), which leads to ecological damage and huge economic loss in aquaculture industries. Cyclophilins (CYPs) belong to the immunophilin superfamily, and they may play a role in the survival mechanisms of the dinoflagellate in stress environments. In the present study, we identified a novel cyclophilin gene from C. polykrikoides and examined physiological and gene transcriptional responses to biocides copper sulphate (CuSO 4 ) and sodium hypochlorite (NaOCl). The full length of CpCYP was 903 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the polyA tail, comprising a 639 bp ORF, a 117 bp 5'-UTR, and a 147 bp 3'-UTR. Motif and phylogenetic comparisons showed that CpCYP was affiliated to group B of CYP. In biocide stressors, cell counts, chlorophyll a , and photosynthetic efficiency ( Fv / Fm ) of C. polykrikoides were considerably decreased in both exposure time- and dose-dependent manners. In addition, CpCYP gene expression was significantly induced after 24 h exposure to the biocide-treated stress conditions. These results indicate an effect of the biocides on the cell physiology and expression profile of CpCYP , suggesting that the gene may play a role in environmental stress responses.

Acetaminophen is the leading cause of acute liver failure (ALF) in many countries including the United States. Hepatic glucuronidation by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes is the major route of acetaminophen elimination. Reduced glucuronidation may predispose some individuals to acetaminophen-induced ALF, but mechanisms underlying reduced glucuronidation are poorly understood. We hypothesized that specific microRNAs (miRNAs) may reduce UGT1A activity by direct effects on the UGT1A 3'-UTR shared by all UGT1A enzyme transcripts, or by indirect effects on transcription factors regulating UGT1A expression. We performed an unbiased miRNA whole transcriptome association analysis using a bank of human livers with known acetaminophen glucuronidation activities. Of 754 miRNAs evaluated, 9 miRNAs were identified that were significantly overexpressed (p<0.05; >2-fold) in livers with low acetaminophen glucuronidation activities compared with those with high activities. miR-375 showed the highest difference (>10-fold), and was chosen for further mechanistic validation. We demonstrated using in silico analysis and luciferase reporter assays that miR-375 has a unique functional binding site in the 3'-UTR of the aryl hydrocarbon receptor (AhR) gene. Furthermore overexpression of miR-375 in LS180 cells demonstrated significant repression of endogenous AhR protein (by 40%) and mRNA (by 10%), as well as enzyme activity and/or mRNA of AhR regulated enzymes including UGT1A1, UGT1A6, and CYP1A2, without affecting UGT2B7, which is not regulated by AhR. Thus miR-375 is identified as a novel repressor of UGT1A-mediated hepatic acetaminophen glucuronidation through reduced AhR expression, which could predispose some individuals to increased risk for acetaminophen-induced ALF., (Published by Elsevier Inc.)

MicroRNAs (miRNAs), which are a class of small noncoding RNAs, can modulate the expression of many protein-coding genes when an organism is exposed to an environmental chemical. We previously demonstrated that miR-155 was significantly downregulated in adult zebrafish (Danio rerio) in response to fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl) phenyl]-4-[(trifluoromethyl) sulphinyl]-1H-pyrazole-3-carbonitrile) exposure. However, the regulation of this miRNA's predicted target gene cyb561d2, which is a member of the cytochrome b561 (cyt b561) family involved in electron transfer, cell defence, and chemical stress, has not been experimentally validated to date. In this study, we evaluated the effects of fipronil on miR-155 and cyb561d2 in zebrafish. The expression of miR-155 was downregulated, whereas cyb561d2 was upregulated in both mRNA and protein level in a dose-dependent manner upon stimulation of fipronil. The dual luciferase report assay demonstrated that miR-155 interacted with cyb561d2 3'-untranslated regions (3'-UTR). The expression of cyb561d2 was reduced in both mRNA and protein levels when ZF4 cells were transfected with an miR-155 mimic, whereas its expression levels of both mRNA and protein were increased when endogenous miR-155 was inhibited by transfection with an miR-155 inhibitor. The results improved our understanding of molecular mechanism of toxicity upon fipronil exposure, and presents miR-155 as a potential novel toxicological biomarker for chemical exposure. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 877-886, 2016., (© 2014 Wiley Periodicals, Inc.)

Glioblastoma (GBM) is a highly invasive malignant primary brain tumor with neoplastic growth. Despite the progresses made in surgery, chemotherapy, and radiation in recent decade, the prognosis of patients with gliomas remains poor and the average survival time of patients suffering from glioblastoma is still short. As a potential therapy strategy, microRNAs have been considered as new targets for possible cancer treatment. In this study, we found that the miR-92b inhibitors (miR-92b-I) could inhibit the proliferation, migration, invasion, and promote the apoptosis of glioma cells. As a predicted target of miR-92b, phosphatase and tensin homolog (PTEN), also elevated at both mRNA and protein levels. Moreover, the Akt phosphorylation was consistently inhibited. The rescue experiment with miR-92b and PTEN double knockdown resulted in partial reversion of miR-92b-I-induced phenotypes. Taken together, our findings indicated that miR-92b-I could restrain the proliferation, invasion, migration, and stimulate apoptosis of glioma cells by targeting PTEN/Akt signaling pathway. Further investigations will focus on antitumor effect of miR‑92b-I in glioma treatment.

The proliferation of cardiac fibroblasts (CFs) and excessive deposition of extracellular matrix (ECM) are the main pathological characteristics of cardiac fibrosis. In recent years, microRNAs (miRNAs) have been found to be a new kind of regulator in cardiac fibrosis. The purpose of this study was to investigate the role of microRNA-9 (miR-9) in the process of cardiac fibrosis and its mechanism. Treatment of cultured neonatal rat CFs with PDGF-BB or serum suppressed the expression of miR-9. Overexpression of miR-9 obviously inhibited neonatal rat CFs proliferation and collagen production as detected by MTT assays, qRT-PCR, and western blotting. The effects of miR-9 in CFs were abrogated by co-transfection with miR-9 inhibitors. Overexpression of miR-9 reduced the mRNA and protein levels of PDGFR-βand its downstream protein, extracellular signal-regulated kinase (ERK) 1/2. Silencing PDGFR-βby small interfering RNA mimicked the anti-fibrotic action of miR-9, whereas overexpression of PGDFR-β canceled the effect of miR-9 in cultured CFs. Dual-luciferase reporter assays showed that PDGFR-βwas a direct target of miR-9. Overexpression of miR-9 inhibited cardiac fibrosis by targeting PDGFR-β, indicating that miR-9 might play a role in the treatment of cardiac fibrosis.

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

MicroRNAs (miRNA) are small non-coding RNA molecules representing a novel class of endogenous negative regulators of gene expression. MiRNA have the ability to bind to specific regions in the 3'UTR of mRNA and repress gene expression through interaction with the RNA induced silencing complex (RISC). They have now been implicated in the pathophysiology of many kidney diseases, including the onset and progression of tubulointerstitial and glomerulosclerosis and have potential as biomarkers and as novel targets for treatment. The unique feature of miRNAs to target multiple mRNAs defines that targeting a particular miRNA for therapy could have a dramatic effect on the disease process. This review will focus on our current understanding of the role of miRNA in renal diseases, including diabetes, renal fibrosis, IgA nephropathy and explore the miRNA targets which represent the most promising in terms of clinical translation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. While surgery remains the mainstay of treatment for early stage CRC, adjuvant chemotherapy is usually given to reduce the risk of recurrence after colectomy. Overexpression of a multidrug resistance (MDR) transporter ABCG2 in vitro has been shown to cause resistance to 5-fluorouracil (5-FU) and irinotecan, components of the most commonly adopted regimens for treating CRC. Both anticancer drugs are known ABCG2 substrates. An effective way to predict drug response may provide guidance for better cancer treatment. We investigated the effect of ABCG2 dysregulation on cancer cell sensitivity to chemotherapy using pairs of snap-frozen paraffin-embedded archival blocks of human colorectal cancer tissues and their matched non-cancerous colon tissues from CRC patients. In CRC patients responding to chemotherapy, the tumors were found to have remarkable lower ABCG2 expression than the adjacent normal colon tissues. On the contrary, the tumors from patients not responding to 5-FU-based chemotherapy have higher ABCG2 level than the adjacent normal tissues. The high ABCG2 expression in the tumor is associated with the concomitant overexpression of the mRNA binding protein HuR but a low expression of miR-519c because miR-519c is known to target both ABCG2 and HuR. Further investigation in CRC cell lines revealed that the ABCG2 overexpression was caused by an interplay between miR-519c, HuR and the length of the 3' untranslated region (UTR) of ABCG2. These parameters may be further developed as useful biomarkers to predict patient response to adjuvant chemotherapy. Besides being predictive biomarkers, the microRNAs and mRNA binding protein identified may also be potential drug targets for modulating ABCG2 to combat resistance in CRC chemotherapy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Multiple myeloma (MM) is a common and deadly cancer of blood plasma cells. A unique feature of MM is the extremely low somatic mutation rate of the p53 tumor suppressor gene, in sharp contrast with about half of all human cancers where this gene is frequently mutated. Eleven miRNAs have been reported to repress p53 through direct interaction with the 3' untranslated region. The expression of nine of them is higher in MM plasma cells than in healthy donor counterparts, suggesting that miRNA overexpression is responsible for p53 inactivation in MM. Here, we report that the environmental carcinogen benzo[a]pyrene (BaP) upregulated the expression of seven p53-targeting miRNAs (miR-25, miR-15a, miR-16, miR-92, miR-125b, miR-141, and miR-200a), while 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) upregulated two of them (miR-25 and miR-92) in MM cells. The miR-25 promoter was activated by both BaP and TCDD, and this response was mediated by the aryl hydrocarbon receptor (AhR). We screened 727 compounds that inhibit MM cell survival and down-regulate the expression of p53-targeting miRNAs. We found that (-)-epigallocatechin-3-gallate (EGCG), a constituent of green tea and a major component of the botanical drug Polyphenon® E, reduced the expression of four p53-targeting miRNAs, including miR-25, miR-92, miR-141, and miR-200a. Collectively, these data implicate polycyclic aromatic hydrocarbons and AhR in the regulation of p53-targeting miRNAs in MM and identify a potential therapeutic and preventive agent to combat this deadly disease., (© 2014 Wiley Periodicals, Inc.)

Placental growth factor (PlGF) plays an important role in various pathological conditions and diseases such as inflammation, cancer, atherosclerosis and sickle cell disease (SCD). Abnormally high PlGF levels in SCD patients are associated with increased inflammation and pulmonary hypertension (PHT) and reactive airway disease; however, the transcriptional and post-transcriptional mechanisms regulating PlGF expression are not well defined. Herein, we show that treatment of human erythroid cells and colony forming units with erythropoietin (EPO) increased PlGF expression. Our studies showed EPO-mediated activation of HIF-1α led to subsequent binding of HIF-1α to hypoxia response elements (HREs) within the PlGF promoter, as demonstrated by luciferase transcription reporter assays and ChIP analysis of the endogenous gene. Additionally, we showed miR-214 post-transcriptionally regulated the expression of PlGF as demonstrated by luciferase reporter assays using wild-type (wt) and mutant PlGF-3'-UTR constructs. Furthermore, synthesis of miR-214, located in an intron of DNM3 (dynamin 3), was transcriptionally regulated by transcription factors, peroxisome proliferator-activated receptor-α (PPARα) and hypoxia-inducible factor-1α (HIF-1α). These results were corroborated in vivo wherein plasma from SCD patients and lung tissues from sickle mice showed an inverse correlation between PlGF and miR-214 levels. Finally, we observed that miR-214 expression could be induced by fenofibrate, a Food and Drug Administration (FDA) approved PPARα agonist, thus revealing a potential therapeutic approach for reduction in PlGF levels by increasing miR-214 transcription. This strategy has potential clinical implications for several pathological conditions including SCD., (© The Authors Journal compilation © 2015 Biochemical Society.)

Prenatal exposure to diethylstilbestrol (DES) is known to cause an increased susceptibility to a wide array of clinical disorders in humans. Previous studies from our laboratory demonstrated that prenatal exposure to DES induces thymic atrophy and apoptosis in the thymus. In the current study, we investigated if such effects on the thymus result from alterations in the expression of microRNA (miR). To that end, pregnant C57BL/6 mice who were exposed to DES and miR profiles in thymocytes of both the mother and fetuses on postnatal day 3 (gestation day 17) were studied. Of the 609 mouse miRs examined, we noted 59 altered miRs that were common for both mothers and fetuses, whereas 107 altered miRs were specific to mothers only and 101 altered miRs were specific to fetuses only. Upon further analyses in the fetuses, we observed that DES-mediated changes in miR expression may regulate genes involved in important functions, such as apoptosis, autophagy, toxicity, and cancer. Of the miRs that showed decreased expression following DES treatment, miR-18b and miR-23a were found to possess complementary sequences and binding affinity for 3' untranslated regions of the Fas ligand (FasL) and Fas, respectively. Transfection studies confirmed that DES-mediated downregulation of miR-18b and miR-23a led to increased FasL and Fas expression. These data demonstrated that prenatal DES exposure can cause alterations in miRs, leading to changes in the gene expression, specifically, miR-mediated increased expression in FasL and Fas causing apoptosis and thymic atrophy., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.

Background: Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator in cancer cell survival, epithelial-mesenchymal transition, and tumorigenesis. Inhibition of EZH2 has become a promising therapeutic option for various human malignancies. Previously, we demonstrated that the EZH2/miR-30d/karyopherin (importin) beta 1 (KPNB1) signaling pathway is critical for malignant peripheral nerve sheath tumor (MPNST) cell survival in vitro and for tumorigenesis in vivo. Here, we sought to determine the antitumor effects of pharmacological inhibition of EZH2 on MPNST in vitro and in vivo., Methods: We investigated the effects of an EZH2 inhibitor, 3-deazaneplanocin A (DZNep), on MPNST cell cycle, survival and apoptosis in vitro and on MPNST xenograft tumor growth in vivo., Results: We found that DZNep treatment impaired MPNST cell viability and proliferation by inducing apoptosis and cell cycle arrest in vitro. Consistently, DZNep treatment also reduced EZH2 and KPNB1 protein levels and upregulated miR-30d expression in MPNST cells. Intraperitoneal administration of DZNep significantly suppressed MPNST tumor initiation and growth rates in a MPNST xenograft mouse model. Immunoblot and immunohistochemical analyses showed that DZNep downregulated EZH2/KPNB1 signaling in vivo, thereby inhibiting MPNST tumor cell proliferation, and induced cell death. We also found that EZH2 inhibited expression of another miR-30 family member, miR-30a, in MPNST cells. Similar to miR-30d, miR-30a inhibited KPNB1 by targeting the KPNB1 3' untranslated region in MPNST cells. Our data also showed that EZH2 suppressed miR-200b expression and induced epithelial-mesenchymal transition in MPNST cells., Conclusion: These findings demonstrated that DZNep, an inhibitor of S-adenosyl-methionine-dependent methyltransferase, suppressed EZH2/miR-30a,d/KPNB1 signaling and blocked MPNST tumor cell growth and survival in vitro and in vivo. More importantly, our study indicated that pharmacological interference of EZH2 is a potential therapeutic approach for MPNST.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3' untranslated region (3' UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3' UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3' UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3' UTR.

Background: Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide. A major cause for the failure of cancer therapy is the development of chemoresistance. Although progress has been made in the study of the mechanisms underlying cancer cells resistance, little is known about the role of microRNAs (miRNAs) in cancer therapy resistance., Methods and Results: Fifteen miRNAs, including 6 up-regulated miRNAs (> 2.0-fold) and 9 down-regulated miRNAs (< 0.5-fold) were differentially expressed in 5-fluorouracil-resistant and their parental cell-lines (HepG2, HepG2/5-FU) by miRNA microarrays. Microarray results were confirmed by validating quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Up-regulation of miR-141 expression resulted in a significant inhibition of 5-FU-mediated cytotoxicity and apoptosis in various hepatocellular carcinoma cells-lines. Mechanically, miR-141 promoted Kelch-like ECH-associated protein 1 (Keap1) mRNA degradation by directly targeting the Keap1 3'untranslated region (3'UTR). Treatment with miR-141 mimics in parental HepG2 cells, restored miR-141 expression and reduced Keap1 levels, thereby resulting in erythroid transcription factor NFE2-L2 (Nrf2) nuclear translocation, activation of Nrf2-dependent HO-1 gene transcription, and subsequent enhancement in 5-FU resistance. Conversely, restoring the expression of Keap1 partly recovered 5-FU sensitivity by counteracting miR-141-mediated 5-FU resistance., Conclusion: Our study showed that miR-141 plays a key role in 5-FU resistance by down-regulating Keap1 expression, thereby reactivating the Nrf2-dependent antioxidant pathway, which may serve as a potential target for overcoming 5-FU resistance in hepatocellular carcinoma cells., (© 2015 S. Karger AG, Basel.)

Aims: To characterize the effects of long-term β-adrenergic receptor stimulation on Rem protein and mRNA expression in rat heart and possible involvement of miR-132., Methods: Adult rats were treated with isoproterenol (ISO, 150 µg.kg.h(-1)) for 2 d and Rem, miR-132, and α1c (the principal subunit of Cav1.2 channels) were measured at protein and mRNA levels with western blot and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiments, respectively. Ca(2+) currents and intracellular Ca(2+) signals were evaluated in isolated cardiomyocytes., Results: Systemic administration of ISO led to decreases in Rem protein and mRNA levels (down to 49%). Furthermore, levels of the microRNAs (miRs) miR-132 and miR-214 were upregulated 5- and 9-fold, respectively. Transfection of miR-132, but not miR-214, into HEK293 cells reduced the expression of a luciferase reporter gene controlled by a conserved 3´-untranslated region (UTR) of Rem by half. Chronic ISO administration also led to a 25% decrease in the amplitude of peak L-type Ca(2+) currents, a 40% decrease in α1c subunit protein abundance at the membrane level, and a 60% decrease in expression of α1c channel subunit mRNA., Conclusions: These results suggest that Rem expression is down-regulated posttranscriptionally by miR-132 in response to long-term activation of β-adrenergic signaling, but this down-regulation does not produce a larger Ca(2+) influx through Cav1.2 channels.

Initially identified as an occasional and peculiar mode of gene regulation in eukaryotes, RNA-binding protein-mediated post-transcriptional control of gene expression has emerged, over the last two decades, as a major contributor in the control of gene expression. A large variety of RNA-binding proteins (RBPs) allows the recognition of very diverse messenger RNA sequences and participates in the regulation of basically all cellular processes. Nevertheless, the rapid outcome of post-transcriptional regulations on the level of gene expression has favored the expansion of this type of regulation in cellular processes prone to rapid and frequent modulations such as the control of the inflammatory response. At the molecular level, the 3'untranslated region (3'UTR) of mRNA is a favored site of RBP recruitment. RBPs binding to these regions control gene expression through two major modes of regulation, namely mRNA decay and modulation of translational activity. Recent progresses suggest that these two mechanisms are often interdependent and might result one from the other. Therefore, different RBPs binding distinct RNA subsets could share similar modes of action at the molecular level. RBPs are frequent targets of post-translational modifications, thereby disclosing numerous possibilities for pharmacological interventions. However, redundancies of the transduction pathways controlling these modifications have limited the perspectives to define RBPs as new therapeutic targets. Through the analysis of several examples of RBPs binding to 3'untranslated region of mRNA, we present here recent progress and perspectives regarding this rapidly evolving field of molecular biology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

TGFβR1 plays an important role in TGF-β signaling transduction and serves as a tumor suppressor. Our previous studies show that reduced expression of TGFβR1 is common in non-small cell lung cancer (NSCLC) and TGFβR1 variants confer risk of NSCLC. However, the epigenetic mechanisms underlying the role of TGFβR1 in NSCLC carcinogenesis are still elusive. We investigated the function and regulation of TGF-β signaling-based miRNAs in NSCLC. Computational algorithms predicted that the 3'-untranslated region (3'-UTR) of TGFβR1 is a target of miR-142-3p. Here a luciferase reporter assay confirmed that miR-142-3p can directly bind to 3'-UTR of TGFβR1. Overexpression of miR-142-3p in NSCLC A549 cells suppressed expression of TGFβR1 mRNA and protein, while knockdown of endogenous miR-142-3p led to increased expression of TGFβR1. On TGF-β1 stimulation, stable overexpression of miR-142-3p attenuated phosphorylation of SMAD3, an indispensable downstream effector in canonical TGF-β/Smad signaling, via repression of TGFβR1 in A549 cells. Furthermore, miR-142-3p-mediated down-regulation of TGFβR1 weakened TGF-β-induced growth inhibition effect, and this effect was reversed by stable knockdown of endogenous miR-142-3p in A549 cells. In NSCLC tissues, miR-142-3p expression was increased and inversely correlated with TGFβR1 expression. These data demonstrate that miR-142-3p influences the proliferation of NSCLC cells through repression of TGFβR1., (© FASEB.)

miR-30d has been observed to be significantly down-regulated in human anaplastic thyroid carcinoma (ATC), and is believed to be an important event in thyroid cell transformation. In this study, we found that miR-30d has a critical role in modulating sensitivity of ATC cells to cisplatin, a commonly used chemotherapeutic drug for treatment of this neoplasm. Using a mimic of miR-30d, we demonstrated that miR-30d could negatively regulate the expression of beclin 1, a key autophagy gene, leading to suppression of the cisplatin-activated autophagic response that protects ATC cells from apoptosis. A reporter gene assay demonstrated that the binding sequences of miR-30d in the beclin 1-3' UTR was the region required for the inhibition of beclin 1 expression by this miRNA. We further showed that inhibition of the beclin 1-mediated autophagy by the miR-30d mimic sensitized ATC cells to cisplatin both in vitro (cell culture) and in vivo (animal xenograft model). These results suggest that dysregulation of miR-30d in ATC cells is responsible for the insensitivity to cisplatin by promoting autophagic survival. Thus, miR-30d may be exploited as a potential target for therapeutic intervention in the treatment of ATC., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most common cause of lung cancer death. Currently, the epidermal growth factor receptor inhibitor gefitinib is used for its treatment; however, drug resistance is a major obstacle. Expression of Met has been associated with both primary and acquired resistance to gefitinib, but the mechanisms regulating its expression are not fully understood. Recently, miRNAs such as miR-130a have been shown to play a role in gefitinib resistance, but importance in NSCLC and relationships with Met have not been fully explored. Here we show that miR-130a is over-expressed in gefitinib- sensitive NSCLC cell lines, but is low in gefitinib-resistant NSCLC cell lines. Moreover, miR-130a expression was negatively correlated with that of Met. Further analysis revealed that over-expression of miR-130a increased cell apoptosis and inhibited proliferation of NSCLC cells treated with gefitinib, whereas lowering the expression of miR-130a decreased cell apoptosis and promoted cell proliferation after treatment with gefitinib in both gefitinib-sensitive and -resistant NSCLC cell lines, suggesting that miR-130a overcomes gefitinib resistance. We also demonstrated that miR-130a binds to the 3'-UTR of Met and significantly suppresses its expression. Finally, our results showed that over-expressing Met could "rescue" the functions of miR-130a regarding cell apoptosis and proliferation after cells are treated with gefitinib. These findings indicate that the miR-130a/Met axis plays an important role in gefitinib resistance in NSCLC. Thus, the miR-130a/Met axis may be an effective therapeutic target in gefitinib-resistant lung cancer patients.

Arterial calcification is a key pathologic component of vascular diseases such as atherosclerosis, coronary artery disease, and peripheral vascular disease. A hallmark of this pathological process is the phenotypic transition of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Several studies have demonstrated that microRNAs (miRNAs) regulate osteoblast differentiation, but it is unclear whether miRNAs also regulate VSMC-mediated arterial calcification. In the present study, we sought to characterize the role of miR-133a in regulating VSMC-mediated arterial calcification. Northern blotting analysis of VSMCs treated with β-glycerophosphate demonstrated that miR-133a was significantly decreased during osteogenic differentiation. Overexpression of miR-133a inhibited VSMC transdifferentiation into osteoblast-like cells as evidenced by a decrease in alkaline phosphatase activity, osteocalcin secretion, Runx2 expression, and mineralized nodule formation. Conversely, the knockdown of miR-133a using an miR-133a inhibitor promoted osteogenic differentiation of VSMCs by increasing alkaline phosphatase activity, osteocalcin secretion, and Runx2 expression. Runx2 was identified as a direct target of miR-133a by a cotransfection experiment in VSMCs with luciferase reporter plasmids containing wild-type or mutant 3'-untranslated region sequences of Runx2. Furthermore, the pro-osteogenic effects of miR-133a inhibitor were abrogated in Runx2-knockdown cells, and the inhibition of osteogenic differentiation by pre-miR-133a was reversed by overexpression of Runx2, providing functional evidence that the effects of miR-133a in osteogenic differentiation were mediated by targeting Runx2. These results demonstrate that miR-133a is a key negative regulator of the osteogenic differentiation of VSMCs.

miR-21 can act as an oncogene. MSH2 has been reported that it involved in the DNA mismatch repair (MMR) system and overexpression of MSH2 can induce cell apoptosis. We predicted that MSH2-3'-untranslated region (3'-UTR) was targeted by miR-21 using microRNA analysis softwares. To further explore the roles of miR-21 and MSH2 in A549 cells, we constructed pcDNA-GFP-msh-UTR vector (including MSH2-3'-UTR) to transfect A549 cells with miR-21, GFP positive cells were estimated under a fluorescence microscopy and by flow cytometry. We found miR-21 could obviously downregulate the expression of MSH2, which was further proved by western blotting. Moreover, we treated A549 cells with cisplatin and found that cisplatin could inhibit A549 cell growth in vitro and in vivo. We also found that cisplatin could downregulate miR-21 expression, while increase MSH2 expression in A549 cells. Our results demonstrated that cisplatin could upregulate the expression of MSH2 through downregulating miR-21 to inhibit A549 cell proliferation, which provides new gene targets for drug design or cancer therary., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

While many mechanisms have been proposed for microRNAs (miRNAs) function, most ultimately cause message degradation. A view has emerged that miRNAs silence gene expression by promoting the association of mRNA decay factors. Recent research results, however, suggest that in both zebrafish and fruit fly, translational inhibition is the initiating event of miRNA-mediated gene silencing.

Recently, we have reported tissue- and stage-specific expression of miR-195 in human hepatocellular carcinoma cells and so far, not many reports discuss the function of this microRNA (miRNA). Expression profiling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miR-195 in BEL-7402/5-FU cells. Western blots were performed to determine protein levels of LATS2, P53 and CDK2. MTT analysed the cell proliferation activity. Flow cytometry were performed to determine apoptosis rate. Up-regulation of miR-195 increased expression of LATS2 and increased apoptosis of HCC cells, while Anti-miR-195 treatment inhibited expression of LATS2. miR-195 over-expression inhibited the luciferase activity of a LATS2 3' untranslated region-based reporter construct in BEL-7402/5-FU cells. These results indicate that miR-195 could increase cell apoptosis by targeting LATS2 in hepatocellular carcinoma cells.

Histone deacetylases (HDACs) play a crucial role in tumorigenesis. Over-expression of HDACs has been reported in lung cancer. The mechanism of highly expressed HDAC1 in lung cancer has yet not been determined. In the present study, we showed that miR-449a/b regulates HDAC1 by directly binding with the 3' untranslated region of the HDAC1. The expression of miR-449a/b was down-regulated and the expression of HDAC1 was up-regulated in primary lung cancer. The down expression of miR-449a/b might be one mechanism for over-expression of HDAC1 in lung cancer. miR-449a/b inhibited cell growth and anchorage-independent growth. Furthermore, co-treatment with miR-449a and HDAC inhibitors had a significant growth reduction compared with HDAC inhibitor mono-treatment. These results suggest that miR-449a/b may have a tumor suppressor function and might be a potential therapeutic candidate in patients with primary lung cancer., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs), which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i) the Tet Repressor protein (TetR) genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii) a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, has been implicated as critical regulatory molecules in many cardiovascular diseases, including ischemia/reperfusion induced cardiac injury. Here, we report microRNA-145, a tumor suppressor miRNA, can protect cardiomyocytes from hydrogen peroxide H₂O₂-induced apoptosis through targeting the mitochondrial pathway. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-145 in either ischemia/reperfused mice myocardial tissues or H₂O₂-treated neonatal rat ventricle myocytes (NRVMs) was markedly down-regulated. Over-expression of miR-145 significantly inhibited the H₂O₂-induced cellular apoptosis, ROS production, mitochondrial structure disruption as well as the activation of key signaling proteins in mitochondrial apoptotic pathway. These protective effects of miR-145 were abrogated by over-expression of Bnip3, an initiation factor of the mitochondrial apoptotic pathway in cardiomyocytes. Finally, we utilized both luciferase reporter assay and western blot analysis to identify Bnip3 as a direct target of miR-145. Our results suggest miR-145 plays an important role in regulating mitochondrial apoptotic pathway in heart challenged with oxidative stress. MiR-145 may represent a potential therapeutic target for treatment of oxidative stress-associated cardiovascular diseases, such as myocardial ischemia/reperfusion injury.

To explore the possible microRNAs (miRNAs) in the TP53 pathway and their roles in A549 cell death induced by cisplatin, the miRNAs relative to 3'-untranslated region (3'-UTR) of TP53 were predicted by microRNA analysis softwares, which showed that TP53 expression might be targeted by miR-98, miR-453 and miR-485. Then, GFP was used as a reporter gene to reflect whether the 3'-UTR of TP53 was targeted by the predicted miRNAs. After pcDNA-GFP-UTR was constructed, the GFP expression was estimated in A549 cells by the examination of fluorescence microscopy and flow cytometry. The intensity of fluorescence in the miR-98 and miR-453 groups decreased significantly compared with the control group. The percentage of positive GFP cells in miR-98 and miR-453 groups were 30.24% and 32.58%, respectively, much lower than that of NC group (41.86%). The TP53 expression was inhibited after transfection with miR-98/miR-453 by western blot. As the factors in TP53 pathway, Bcl-2 expression was found to be enhanced, and the expression of miR-34a-c was decreased in A549 cells after miR-98/miR-453 treatment. Moreover, the expression of miR-98 and Bcl-2 was decreased, while miR-34a-c and TP53 was increased after A549 treated with cisplatin. Our study demonstrated that miR-98 and miR-453 down-regulated TP53 expression by targeting the 3'-UTR of TP53, and that cisplatin might inhibit A549 cell growth by miR-98 regulating TP53 pathway. Our results indicated that TP53 relevant miRNAs might be the new targets for gene therapy or new drug design., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The aim of this study was to investigate the effect of microRNA-20a on pancreatic carcinoma cell proliferation and invasion and to find a new effective treatment strategy for pancreatic carcinoma. MicroRNA-20a expression was determined in 10 matched normal pancreatic tissues and pancreatic carcinoma by in situ hybridization. Quantitative real-time RT-PCR was used to evaluate the expression of microRNA-20a in two pancreatic carcinoma cell lines (BxPC-3 and Panc-1) and immortal human pancreatic duct epithelial cell line H6C7. Proliferation and invasion capacity were analyzed for the cells with lentivirus-mediated overexpression of microRNA-20a both in vitro and in vivo. In addition, the regulation of signal transducer and activator of transcription proteins 3 (Stat3) by microRNA-20a was determined to elucidate the underlying mechanisms. The pancreatic cancer cell lines (Panc-1 and BxPC-3) stably overexpressing microRNA-20a showed reduced proliferation and invasion capacity in vitro and in vivo, compared with parental cells or cells transfected with a control vector. Furthermore, we found that microRNA-20a negatively regulated Stat3 protein expression in a dose-dependent manner without changing the Stat3 mRNA level and decreased the activity of a luciferase reporter construct containing the Stat3 3'-untranslated region. These results show that microRNA-20a regulates Stat3 at the post-transcriptional level, resulting in inhibition of cell proliferation and invasion of pancreatic carcinoma. It may open a new perspective for the development of effective gene therapy for pancreatic carcinoma.

Histone deacetylase inhibitors (HDACIs) are promising anti-tumor agents that selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Fundamentally, HDACIs are proposed to function by activating the transcription of genes, including the potent cyclin dependent kinase inhibitor p21(WAF1). However, HDACIs primarily increase p21(WAF1) expression at the post-transcriptional level in HepG2 cells, implying that these anti-tumor agents regulate genes at multiple levels. Here, two novel cis-acting elements in the 3' untranslated region (UTR) of p21(WAF1) are identified that control the ability of HDACIs to induce p21(WAF1) mRNA stabilization. Collectively, these studies highlight the complexity of HDACIs in gene regulation., (Copyright © 2010 Elsevier Inc. All rights reserved.)