Different microneedle shapes, such as cylindrical, conic or lancet-like, for specific applications such as micro-indentation or drug delivery, are demonstrated.In this work, two procedures for fabrication of polymeric microneedles based on direct photolithography, without any etching or molding process, are reported. Polyethylene glycol (average molecular weight 250 Da), casted into a silicone vessel and exposed to ultraviolet light (365 nm) through a mask, cross-links when added by a commercial photocatalyzer. By changing the position of the microneedles support with respect to the vessel, different shapes and lengths can be achieved. Microneedles from a hundred microns up to two millimeters have been obtained just tuning the radiation dose, by changing the exposure time (5–15 s) and/or the power density (9–18 mW/cm By exploiting standard technologies and facilities in the fabrication of micro and nano electro mechanical systems, a new generation of biomedical devices is currently realized and continuously developed. Physical and chemical characteristics of these devices can be finely tuned on a very small scale, down to few nanometers, gaining popularity in biomedicine field over the last decade ,2. MicroHydrogels are attractive materials in fabrication of biodevices, since a hydrated gel provides an excellent matrix for encapsulation of functional biological molecules, such as enzymes and peptides ,10,15,16Poly(ethylene glycol) diacrylate (PEGDA) is a biologically inert polymer that rapidly hardens at room temperature in presence of a photo-initiator on exposure to ultraviolet (UV) light, so that it is possible to fabricate PEGDA MNs by standard photolithographic process. PEGDA is hydrophilic, elastic and can be customized to include a variety of biological molecules or inorganic nanoparticles ,17,18,19Generic photolithographic processes in microelectronic technology require that a substrate is covered by spin-coating a liquid photosensitive polymer, called photoresist, which is later hardened by a thermal process. Then, the photoresist is exposed to ultraviolet radiation through a mask, which reproduces the design of the structure to be realized. In our case, the sketch on the mask is the array of circles that constitute the base of needles. UV light creates or destructs “cross-link” bonds of the polymer, in case the resist is “negative” or “positive”, respectively. In this way, the solubility of the exposed resist is drastically different from that in the shade of mask. By dipping the sample in the developing solution, the portion of the photoresist exposed  is removed. As the last step, the so-called etching step, the drawing made of photoresist is then reproduced on the substrate with wet or dry chemical attack .et al. [Kochhar et al. , propose©  has been used just like an ordinary photoresist and the etching step was not longer required to transfer the design on the substrate. The present photolithographic approach to fabricate PEGDA MNs, using microelectronic fabrication facility and skills, allows the realization of MNs with different shape, length and tip by changing the exposure parameters and the position of the support. The processes provide to a wide range of MN types, useful either for epidermis or dermis targeting. In fact, the needle morphological and dimensional features can be decided by adjusting the exposure parameters, depending on the specific application.In this work, we proposed two procedures (Method 1 and Method 2 in the following) of direct photolithography, where the mixture of PEGDA and DAROCURMn = 250, Sigma Aldrich, St. Louis, MO, USA) mixed with 2-Hydroxy-2-methyl-1-phenyl-propan-1-one  at 2% (v/v). The DAROCUR© is a liquid photo-initiator which is used to initiate the photo-polymerization of chemically unsaturated pre-polymer solution [All chemicals are commercially available and used as received. We use a solution of PEGDA  Q83  is a hard sheet of plastic-like acetate in form of a transparent sheet with thickness of 125 μm.© has been used as photoresist and the etching step was not longer required to transfer the design on the substrate  of direct photolithography, where the mixture of PEGDA and DAROCURi.e., it is both a container for the liquid resist and a spacer able to define the height of the needles. The exposure to UV light was made by a mask-aligner with soft contact or in close proximity to prevent contamination of the mask. Finally, samples were developed in deionized water and dried with nitrogen.In this configuration, sketched in Different deposition methods of the liquid PEGDA in several processes have been exploited in order to improve the length and the shape of MNs.In this case, a quartz layer was on top of the vessel and was used as substrate for the hardened needles, once reversed b. The bai.e., the energy density, has been studied by changing the power density of the UV source  and the exposure time . Moreover, both the arrays “A” and “B” have been investigated.The dependence of MNs height and shape on the dose of radiation, For MNs higher than 300 μm, the length was measured by image analysis. The average height of the MNs arrays was evaluated by images captured by the camera of a water contact angle analyzer .Some MN has been removed by the quartz substrate and measured aside. Then, the image has been performed and analyzed by Leica DM6000 M microscope  equipped by Leica DFC 280 digital camera system (Leica).Micro and nano features of MNs, such as surface roughness and curvature radius of the tip, have been evaluated from images by a scanning electron microscopy (SEM) and by the optical microscope.SEM images were performed at 5 kV accelerating voltage and 30 μm wide aperture by a field emission scanning electron microscope . Secondary emission detector has been used. The polymeric MNs were been gold coated to improve the conductivity and, thus, the definition of the SEM images. In order to evaluate the porous structure, the gold has been partially removed by a single MN.vs. distance curve, which is a plot of the force between the AFM tip and the sample as function of their relative distance . In particular, the indentation hardness of the MN tip has been compared with the indentation hardness of a Parafilm®  layer , used as artificial skin as reported in [® have been proved to be very close to that of pigskin. Moreover, penetration test have been performed by using eight Parafilm® layers folded on their self; images of the first layer have been captured both at optical microscope and by the camera of the WAC.Mechanical properties of PEGDA based MNs have been characterized by measuring the indentation hardness of the MN tip by means of force/distance (F/D) spectroscopy tool of the atomic force microscope . The F/D spectroscopy tool supports the acquisition of force orted in , where mThe water content of MNs has been estimated by thermogravimetric analysis (TGA) in order to evaluate the polymer decomposition, as also reported in ref. . The theThe samples have been immersed in Phosphate Buffer PBS  at 37 °C. At fixed times , samples have been recovered and analyzed after removing of water excess.The average water loss was calculated as the loss in weight at 100 °C on three samples at each fixed time.v/v of PEGDA crosslinked, and the excess of unreacted product was removed by water rinsing. The MNs, fabricated by both of the two methods described here, are also hydrophilic in physiological medium (PBS at 37 °C).PEGDA is well known in literature to be a polymer in nature, characterized by hydrophilic behavior even after polymerization, and capability to mimic the role of extracellular matrix of living tissues ,25. We c2) and the exposure time (1000 s) in the mask aligner instrument. Results are summarized in Preliminary investigations were focused on testing two different depositions of PEGDA on the substrate, namely by casting it in a silicone vessel or directly spinning it, once fixed the power density of the lamp . The height of MNs ranged between 1200 and 2200 μm and increases as the exposure time increases, and then the dose increases. MNs fabricated with array A (400 μm diameter) were higher than MNs by array B (250 μm) for equal exposure time. However, the “lancet” shape was demonstrated for both arrays, even with a slight increasing of the exposure time. The effect can be explained by considering that the cross-linked PEGDA is denser than the unexposed one, so that also its refractive index is higher. The ultraviolet light, coming from the mask aligner lamp, is thus confined inside the cross-linked PEGDA , so that the light intensity is higher than elsewhere inside the forming MNs. In this way, a variety of MNs shapes and lengths can be finely tuned by changing the process parameters, In In order to prove the fabrication of smaller MNs, power radiation of the mask aligner UV lamp, at a fixed exposure time (7.5 s), has been decreased. Results are reported in 2 gave the same result in term of MN length  that implies a sort of saturation effect, which limits this kind of fabrication process.In The fabrication procedure optimized in Method 2 allows the production of sharp MNs, which can be useful for skin penetration or indentation in sensing applications ,27, and Fabrication processes were firstly developed on quartz support, but same procedures were repeated using a layer of PEGDA covering the quartz surface, in order to fabricate the MNs array on a flexible support. In the photograph reported in ® layers, used as artificial skin very similar to pig derma, as reported in ref. [® layer (a) compared with the indentation hardness of the central point of a single tip (b). The force spectroscopy plot shows a greater slope, and thus a greater hardness of the MN tip with respect to the artificial skin. This result is essential in quantifying the possibility of a MN in tissue penetration [Since MNs penetration into the skin is a major issue, mechanical properties of MNs have been tested with Parafilm in ref. . Figure etration .® layer. The penetration tests, as described in ref. , have be® layer is 160 μm thick, the MN 1340 μm long penetrates at least 640 μm, proving a good penetration strength. Shorter MNs gave better results in Parafilm® penetration, whereas higher MNs tend to bend on themselves without breaking (data not shown here).In We also investigated the capability of the PEGDA-MNs to properly swell on exposure to water solution. The water content in MNs was estimated by thermogravimetric analysis (TGA).Samples were immersed in PBS solution 10 mM, pH 7.4, at 37 °C. At fixed times , samples were dried and analyzed.As shown in vs. 92.1° tilt angles before and after immersion, respectively. This result confirmed that the PEGDA based MNs demonstrated a great stability in time as well as in physiologic environment, and could be exploited in continuous monitoring application.The water absorption caused a weak deformation of MNs shape, as it can be seen in et al. [in vivo” experiments. However, the proposed MNs are based on strongly cross-linked PEGDA (which means very low water content), as proved by the small water content after 144 h in buffer PBS at 37 °C. Than, the reactivity of PEGDA in proposed MN is lower than in water solution.Finally, any appreciable degradation of MNs array in terms of mechanical properties after a period of 3–5 days could not be found. In agreement with our results, Browning et al.  referredIn this work, we exploited two similar fabrication procedures (Method 1 and Method 2) for producing polymeric MNs arrays on both hard and flexible supports. MNs were made of PEGDA, that allows casting and direct polymerization, thus eliminating etching step in the fabrication process and drastically decreasing production cost.Method 1 (support at bottom) enables the fabrication of MNs with cylindrical shape and heights up to 150 μm. Method 2 (support at the top) enables conical and lancet shapes with tunable highness in the range from 150 to 2240 μm, by simply changing the dose of exposure radiation in terms of time and power. Method 2 resulted faster than Method 1, since exposure time was less by two orders of magnitude. Finally, the presented methods can be easily integrated in a microelectronic fabrication process, and due to their properties this kind of MNs can be the perfect interface between the patient body and a microelectronic device for health monitoring.


Furthermore, miR-373-3p directly targeted and suppressed Dickkopf-1 (DKK1), a negative regulator of the Wnt/β-catenin signaling cascade. These results demonstrate that, by directly targeting DKK1, miR-373-3p constitutively activated Wnt/β-catenin signaling, thus promoting the EMT-induced metastasis of TSCC. Taken together, our findings reveal a new regulatory mechanism for miR-373-3p and suggest that miR-373-3p might be a potential target in TSCC therapy.MicroRNAs (miRNAs) regulate gene expression and at the same time mediate tumorigenesis. miR-373-3p has diverse effects in tumors, but its role in tongue squamous cell carcinoma (TSCC) remains unknown. The purpose of this study is to determine the function of miR-373-3p in the progression of TSCC. Our results brought to light that miR-373-3p is markedly upregulated in clinical TSCC tissues compared with paired adjacent normal tissues and has significant correlation with a more aggressive TSCC phenotype in patients. Gain-of-function and loss-of-function studies revealed that ectopic miR-373-3p overexpression promoted the metastasis of TSCC cells. Notably, Wnt/ Tongue squamous cell carcinoma (TSCC) is the most common cancer of the oral cavity and is a very aggressive disease . Over thβ-catenin signaling pathway is an established regulator of the EMT due to its involvement in the maintenance of epithelial integrity and tight adherens junctions [β-catenin is stabilized and allowed to accumulate. Activated β-catenin dissociates from E-cadherin, which disassembles adherens junctions and then enters the nucleus to activate the expression of target genes, which accounts for the upregulation of EMT biomarkers and an ultimately unfavorable cancer prognosis [β-catenin pathway, functions as a negative regulator of Wnt signaling [β-catenin signaling pathway is considered an important regulator of cancer cell metastasis; therefore, more in-depth researches of the mechanisms that regulate the Wnt/β-catenin pathway may provide a new perspective for more effective TSCC therapies.The Wnt/unctions . Mutatiounctions . Upon Wnrognosis , 8. Dickignaling . It has ignaling –11. AccomiRNAs are a class of small noncoding RNAs that are approximately 22 nucleotides in length. Typically, miRNAs bind to the complementary sequence in the 3′-untranslated regions (3′-UTRs) of mRNAs, leading to the degradation or inhibited translation of the targeted mRNAs . Bioinfoβ-catenin signaling in TSCC and to discern the relevant molecular mechanisms. Our data show that miR-373-3p is upregulated in tissues isolated from patients with TSCC. Furthermore, miR-373-3p activates the Wnt/β-catenin pathway by directly targeting DKK1 to consequently promote EMT and metastasis of TSCC.In this study, we sought to elucidate the effect of miR-373-3p on Wnt/Fresh TSCC tissue samples from TSCC patients and their adjacent nonneoplastic tissue samples were collected with informed consent under the institutional board-approved protocols from the Hospital of Stomatology, Sun Yat-sen University . The samples had been clinically and histopathologically diagnosed according to the World Health Organization criteria. Tumor and noncancerous tissues were confirmed histologically by hematoxylin and eosin staining. This study was approved by the institutional research ethics committee of the Hospital of Stomatology, Sun Yat-sen University. TSCC cell lines SCC-9, SCC-15, SCC-25, UM1, and UM2 were routinely maintained in DMEM  supplemented with 10% fetal bovine serum .5 cells per well) the day before transfection. Cells were then transfected with the firefly luciferase reporter plasmid including either the wild-type or mutant 3′-UTR of DKK1 (50 ng per well) and the pRL-TK Renilla luciferase reporter (10 ng per well). The cells were then transfected with miR-373-3p mimics or antagomiR-373-3p (50 nM) using Lipofectamine 2000 (Invitrogen). Luciferase and Renilla signals were measured 24 h after transfection using a Dual Luciferase Reporter Assay Kit (Promega).The wild-type 3′-UTR of human DKK1 was subcloned into pmirGLO using SpeI and HindIII cut sites . Mutations of the 3′-UTR sequence were created with a Quick Change Site-Directed Mutagenesis Kit . HEK293T cells were seeded onto 24-well plates (1 × 10β-catenin (siβ-catenin), siRNA-T cell factor 4 (siTCF4), and corresponding control oligonucleotides  were transfected into SCC-9 and UM1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The sense strand sequences of siRNAs were as follows: siDKK1, 5′-GAAUAUGUGUGUCUUCUGAUU-3′; siβ-catenin, 5′-UGGUUGCCUUGCUCAACAA-3′; and siTCF4, 5′-AAGUCCGAGAAAGGAAUCUGA-3′.miR-373-3p mimics, antagomiR-373-3p, siRNA-DKK1 (siDKK1), siRNA-β-Actin and U6 were used to normalize mRNA and miRNA, respectively. The sequences of the primers were as follows: E-cadherin forward, 5′-GACCGGTGCAATCTTCAAA-3′; E-cadherin reverse, 5′-TTGACGCCGAGAGCTACAC-3′; vimentin forward, 5′-TTCTTCCCTGAACCTGAGAGA-3′; vimentin reverse, 5′-GAGTGGGTGTCAACCAGAGG-3′; N-cadherin forward, 5′-GTGCCATfAGCCAAGGGAA-3′; N-cadherin reverse, 5′-GCGTTCCTGTTCCACTCATAGGAG-3′; CK18 forward, 5′-AAGGCCTACAAGCCCAGATT-3′; CK18 reverse, 5′-CACTGTGGTGCTCTCCTCAA-3′; CCND1 forward, 5′-TTCTGCCTTTGATGTTAC-3′; CCND1 reverse, 5′-AGGCTGAATCAATGTCTT-3′; c-Myc forward, 5′-TCAAGAGGCGAACACACAAC-3′; c-Myc reverse, 5′-GGCCTTTTCATTGTTTTCCA-3′; LEF1 forward, 5′-CACTGTAAGTGATGAGGGGG-3′; LEF1 reverse, 5′-TGGATCTCTTTCTCCACCCA-3′; MMP-7 forward, 5′-GCATCTCCTTGAGTTTGGCT-3′; MMP-7 reverse, 5′-GAGCTACAGTGGGAACAGGC-3′; GAPDH forward, 5′-GACTCATGACCACAGTCCATGC-3′; and GAPDH reverse, 5′-AGAGGCAGGGATGATGTTCTG-3′. Relative quantitation was calculated using 2−ΔΔCt method. All experiments were carried out in triplicate.Total miRNA from cultured cells and fresh surgical tongue tissues was isolated using TRIzol (Invitrogen). Detection of miR-373-3p and mRNA was performed as previously described . β-ActinCCK-8 assay was performed to detect cell viability according to a standard method, as previously described .The invasion assay was performed using a transwell chamber consisting of 8 mm membrane filter inserts . Briefly, the upper chamber was coated with Matrigel  and incubated for 24 h. The noninvading cells were gently removed with a soft cotton swab, and the cells that invaded to the bottom chamber were fixed with 4% paraformaldehyde, stained with hematoxylin, photographed, and counted.β-catenin and anti-E-cadherin ; anti-DKK1, anti-cytokeratin 18, anti-N-cadherin, and anti-vimentin ; Anti-Nuclear Matrix Protein p84 ; and anti-α-GAPDH and anti-α-tubulin antibody . Nuclear extracts were prepared using the Nuclear Extraction Kit .Western blotting was performed according to a standard method, as previously described . The fol4) were seeded in triplicate onto 24-well plates and cultured for 24 h. Cells were transfected with 100 ng TOP/FOP reporter luciferase plasmid, pmirGLO-DKK1-3′-UTR, or pmirGLO-DKK1-3′-UTR-mut luciferase plasmids, each in combination with 5 ng of the pRL-TK Renilla plasmid (Promega) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendation. Luciferase and Renilla signals were measured twenty-four hours after transfection using a Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer's protocol.Cells (4 × 10http://targetscan.org/vert_40/); DIANA-mirPath (http://diana.imis.athena-innovation.gr/); and miRanda (http://www.microrna.org/microrna/getGeneForm.do).The following online software programs were used for bioinformatics analysis: TargetScan 4.1 . Statistical differences were determined by the two-tailed Student's To identify the role of miR-373-3p in the development of TSCC, we analyzed the expression of miR-373-3p by quantitative real-time PCR (qRT-PCR) in 63 pairs of TSCC and matched control adjacent normal tissues isolated from patients. Compared to normal tissues, the average expression level of miR-373-3p was significantly increased in tumor tissues . FurtherBy transfecting miR-373-3p mimics into the human TSCC cell lines SCC-9 and UM1, in vitro gain-of-function analyses were performed to explore the effects of miR-373-3p deregulation on the invasiveness of TSCC cells . As showTo further investigate the proinvasive role of miR-373-3p in TSCC, we examined the effect of inhibiting miR-373-3p on the mesenchymal phenotype of SCC-9 and UM1 cells. As expected, miR-373-3p inhibition markedly decreased the EMT Figures  and the β-catenin signaling is frequently activated in TSCC. Since it is one of the most important pathways in maintaining an epithelial cell phenotype and proper cell-cell junctions and tissue homeostasis [β-catenin, whereas silencing miR-373-3p reduced nuclearβ-catenin expression (β-catenin downstream effector genes (β-catenin signaling pathway in TSCC.Wnt/eostasis , we sougpression . Apart for genes . Collectβ-catenin signaling activation in the miR-373-3p-mediated increase of the invasiveness of TSCC cells by silencing either β-catenin or TCF4 in miR-373-3p-overexpressing SCC-9 and UM1 cells. As expected, the stimulation by miR-373-3p of TOP/FOP luciferase reporter activity was impaired by silencing β-catenin or TCF4 (β-catenin or TCF4 abrogated the promotion of TSCC cell invasiveness by miR-373-3p (β-catenin pathway activation is essential for miR-373-3p to promote the invasiveness of TSCC.We further explored the functional significance of Wnt/ or TCF4 . MoreoveR-373-3p . Togetheβ-catenin signaling repressor DKK1 (β-catenin signaling activation. To further validate that DKK1 is a direct target of miR-373-3p, we investigated the relationship between miR-373-3p and DKK1 expression in tissues isolated from TSCC patients and found that DKK1 levels are inversely correlated with miR-373-3p expression levels (β-catenin signaling by directly targeting DKK1.To identify potential targets of miR-373-3p, we used the publicly available algorithms miRanda and TargetScan and identified the Wnt/sor DKK1 . Westernsor DKK1 . Furthersor DKK1 . Howeversor DKK1 . In addisor DKK1 , demonstn levels . Taken tAs hallmarks of aggressive cancer, invasion and metastasis lead to poor prognosis in patients. Exploring the underlying mechanisms of cell migration and invasion is crucial to better understand cancer metastasis and discover potential therapeutic targets. Recent work suggests that miRNAs are important molecular regulators that can modulate cell migration and invasion . Hence, Several researches have suggested that miR-373-3p has a role in multiple cellular processes including proliferation, differentiation, senescence, and apoptosis. These studies also suggested that deregulation is a characteristic of human cancer . miR-373β-catenin, notch, and PI3K/Akt, are aberrantly activated and function in the progression of TSCC [β-catenin signaling functions in TSCC development and progression has been deeply explored [β-catenin signaling pathway, TCF4 and its coactivator β-catenin, are considered to be potent oncogenes in tumor progression [β-catenin are often abnormal in TSCC, which suggests a constitutive activation of Wnt/β-catenin signaling [β-catenin signaling and establish constitutive β-catenin/TCF activation remains unclear. Most importantly, our results established DKK1, a negative regulator of Wnt signaling, as a direct functional effector of miR-373-3p in TSCC, which is in agreement with many studies showing that DKK1 is downregulated in various cancers [β-catenin or TCF4 knockdown suppressed TSCC cell invasion, which phenocopied the effects of miR-373-3p silencing in vitro. These data prove that Wnt signaling plays a role in cell invasion in TSCC and that DKK1 is the direct target site of miR-373-3p.Several signaling pathways, including Wnt/ of TSCC , 7, 8. Agression . Notablyignaling , 29. How cancers –32. So fWe have shown that miR-373-3p is a potent protooncogenic factor in TSCC. miR-373-3p upregulation promotes TSCC invasion by downregulating DKK1, causing Wnt signaling activation, thus inducing EMT. As far as we know, our research has proven to be innovative in demonstrating that the miR-373-3p/DKK1/Wnt signaling axis regulates the invasion of TSCC cells. These findings provide a more profound understanding of the development and progression of TSCC and may have important implications for future therapy against TSCC.


Cre recombinase was injected into the nucleus accumbens and central amygdala to selectively knock down IKKβ in mice genetically engineered with a conditional Ikkb deletion (F/FIkkb). Although IKKβ was inhibited to some extent in astrocytes and microglia, neurons were a primary cellular target. Deletion of IKKβ in either brain region reduced ethanol intake and preference in the continuous access two-bottle choice test without altering the preference for sucrose. Pharmacological and genetic inhibition of IKKβ decreased voluntary ethanol consumption, providing initial support for IKKβ as a potential therapeutic target for alcohol abuse.Proinflammatory pathways in neuronal and non-neuronal cells are implicated in the acute and chronic effects of alcohol exposure in animal models and humans. The nuclear factor-κB (NF-κB) family of DNA transcription factors plays important roles in inflammatory diseases. The kinase IKKβ mediates the phosphorylation and subsequent proteasomal degradation of cytosolic protein inhibitors of NF-κB, leading to activation of NF-κB. The role of IKKβ as a potential regulator of excessive alcohol drinking had not previously been investigated. Based on previous findings that the overactivation of innate immune/inflammatory signaling promotes ethanol consumption, we hypothesized that inhibiting IKKβ would limit/decrease drinking by preventing the activation of NF-κB. We studied the systemic effects of two pharmacological inhibitors of IKKβ, TPCA-1 and sulfasalazine, on ethanol intake using continuous- and limited-access, two-bottle choice drinking tests in C57BL/6J mice. In both tests, TPCA-1 and sulfasalazine reduced ethanol intake and preference without changing total fluid intake or sweet taste preference. A virus expressing  Alcoholism is a devastating disease with few pharmacological treatment options. The disease pathophysiology is unknown, but it is increasingly evident that proinflammatory signaling plays a role. Nuclear factor-κB (NF-κB) is a transcription factor that controls the expression of genes that are involved in inflammation and immunity. IKKβ is a kinase that plays an essential role in regulating the NF-κB signaling pathway. The role of IKKβ in alcohol drinking had not previously been investigated. Our goal was to assess the peripheral and central effects of IKKβ on long-term and binge-like alcohol consumption, and its potential role as a therapeutic target to reduce drinking.Alcohol exposure is known to activate peripheral and central proinflammatory pathways . GenomicNF-κB transcription family members are ubiquitously expressed throughout the body and play important roles in innate/adaptive immunity, cell survival, and inflammation . NF-κB tStudies that have examined the effects of ethanol on IKKβ focused on peripheral effects, such as the exacerbation of pancreatic and hepatic inflammation by chronic ethanol . StudiesCre-mediated IKKβ knockdown in different cell types in the NAc or central amygdala (CeA) on voluntary ethanol consumption. Based on previous studies  to inhibit IKKβ, different brain regions/cell types, and different two-bottle choice (2BC) ethanol drinking paradigms in mice. Two different peripherally acting IKKβ inhibitors, TPCA-1 (2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide) and sulfasalazine, were tested in long-term and binge-like drinking models. TPCA-1 is a selective small-molecule inhibitor of IKKβ . Sulfasa studies , we hypoIkkb gene on a C57BL/6J background . Original breeders were acquired from Casey W. Wright . The C57BL/6J strain was chosen because of its propensity for voluntary ethanol consumption  in temperature- and humidity-controlled rooms. Behavioral testing began when the mice were at least 2 months old. Mice were individually housed at least 2 weeks before beginning the drinking tests. Experiments were conducted in isolated behavioral testing rooms in the Animal Resources Center at The University of Texas at Austin. All experiments were approved by The University of Texas Institutional Animal Care and Use Committee and were conducted in accordance with National Institutes of Health guidelines with regard to the use of animals in research.Pharmacological antagonist studies were conducted in adult male C57BL/6J mice . Genetic knock-down studies were performed in adult male mice with a floxed sumption . Mice wein vivo.Sulfasalazine (Sigma-Aldrich) was injected intraperitoneally, and TPCA-1 (Tocris Bioscience) was administered by mouth. Both drugs were freshly prepared as suspensions in saline solution, with four to five drops of Tween-80, and were injected in a volume of 0.1 ml/10 g of body weight for intraperitoneal administration, and 0.05 ml/10 g of body weight for oral administration. Drugs were administered 30 min prior to ethanol presentation times (see below). Doses of drugs and routes of administration were based on published data that showed anti-inflammatory activity F/FIkkb mice were injected bilaterally (into the NAc or CeA) with either a vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus (LV) expressing Cre recombinase fused to enhanced green fluorescent protein (EGFP) under the control of a cytomegalovirus (CMV) promoter (LV-Cre-EGFP) or an “empty” VSV-G pseudotyped lentiviral vector expressing only the EGFP transgene under a CMV promoter. Mice were anesthetized by isoflurane inhalation, were placed in a stereotaxic apparatus , and were administered a preoperative analgesic (Rimadyl 5 mg/kg). The skull was exposed, and bregma and lambda were visualized with a dissecting microscope. A digitizer attached to the micromanipulator of the stereotaxic apparatus was used to locate coordinates relative to bregma. Burr holes were drilled bilaterally above the injection sites in the skull using a drill equipped with a #75 carbide bit (David Kopf Instruments). The injection sites targeted either the NAc  or the CeA . Injections were performed using a 10 μl microsyringe  and a 30 gauge needle. The needle of the syringe was lowered to the DV coordinate and retracted 0.2 mm. Virus solutions  were injected into each site at a rate of 200 nl/min. After each injection, the syringe was left in place for 5 min before being retracted over a period of 3 min. Incisions were closed with tissue adhesive . Mice were individually housed after surgery and given a 4 week recovery period before starting the ethanol drinking tests.The following three different ethanol-drinking models were used in this study: (1) continuous 24 h 2BC with access to water and ethanol ; (2) 2BC drinking-in-the-dark (DID) with limited 3 h access to 15% ethanol (2BC-DID); and (3) 2BC using ascending concentrations of ethanol solutions .The effects IKKβ antagonists on ethanol intake were measured in adult male C57BL/6J mice in two different drinking paradigms: 2BC with 15% ethanol and 2BC-DID per the protocols previously described . For botF/FIkkb mice using the 24 h 2BC test. Mice treated with either LV-Cre-EGFP or LV-EGFP-Empty were given continuous access to water and ascending concentrations of ethanol solutions  at 2 d intervals , and 24 h intake was calculated. Each concentration was offered for 2 d, and bottle positions were changed daily. The low concentration was presented first, followed by the higher concentrations.One month after completion of the 2BC ethanol test described above, After the completion of behavioral testing, mice were killed by cervical dislocation and decapitated. The brains were quickly removed, flash frozen in liquid nitrogen, and later embedded in Optimal Cutting Temperature (OCT) media in isopentane on dry ice. Brains were then stored at −80°C for future processing. Brains were transferred to a cryostat set at −6°C for at least 1 h before sectioning. Sections (300 μm) were collected from +1.80 to +0.60 mm (AP) relative to bregma and transferred to precooled glass slides on dry ice. Micropunch sampling was performed on a frozen stage (−25°C) using Dual Fluorescent Protein Flashlight (Nightsea), and a mouse stereotaxic atlas to identify the GFP expression and anatomical location of the injection site. Microdissection punches (Stoelting Co.) with an inner diameter of 0.75 mm were used to obtain samples of NAc. This inner diameter fit within the viral spread around the injection site and minimized contamination from other tissue. Punches were taken bilaterally from 4–300 μm sections and were stored at −80°C until RNA extraction. Micropunches were washed with 100% ethanol and RNaseZap (Life Technologies) between each animal. All equipment used to obtain tissue was treated with RNaseZap (Life Technologies) to prevent RNA degradation. Total RNA was extracted using the MagMAX-96 for Microarrays Total RNA Isolation Kit (Life Technologies) according to the manufacturer instructions. RNA yields and purity were assessed using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific). Both the 260:230 and 260:280 ratios were >2.0. RNA quality was determined using the Agilent 2200 TapeStation (Agilent) with RNA integrity numbers averaging >8.0.Ikkb mRNA knockdown, single-stranded cDNA was synthesized from total RNA using the TaqMan High Capacity RNA-to-cDNA Kit (Life Technologies). Following reverse transcription, quantitative real-time PCR was performed in triplicate using TaqMan Gene Expression Assays together with the TaqMan Gene Expression Master Mix (Life Technologies), per the manufacturer instructions. The TaqMan Gene Expression Assays used were Ikbkb (ID #Mm01222247_m1), Tnfa (ID #Mm00443258_m1), Il6 (ID #Mm00446190_m1), and EGFP (ID #Mr04097229_mr). Gapdh  was used as a reference gene, and relative mRNA levels were determined using the 2−▵▵CT method , and data were collected using CFX Manger (Bio-Rad). All genes were normalized to the endogenous housekeeping gene Gapdh and expressed relative to the respective LV-EGFP-Empty control treatment.To verify T method . Gapdh wAnimals were killed, transcardially perfused with PBS and 4% paraformaldehyde (PFA); and brains were harvested, postfixed for 24 h in 4% PFA at 4°C, and cryoprotected for 24 h in 20% sucrose in PBS at 4°C. Brains were placed in molds containing OCT compound (VWR) and frozen in isopentane on dry ice. The brains were equilibrated in a −12 to −14°C cryostat (Thermo Fisher Scientific) for at least 1 h, and coronal sections of 30 µm were taken from the NAc and CeA and placed in sterile PBS.Sections were penetrated with 0.1% Triton X-100 (2 × 10 min at 25°C); washed in PBS (3 × 5 min at 25°C); blocked with 10% goat or donkey serum (30 min at 25°C); treated with 1:250 anti-IKKβ (Millipore), 1:500 anti-NeuN (Santa Cruz Biotechnology), 1:300 anti-GFAP (Santa Cruz Biotechnology), 1:1000 anti-IBA1 (Dako), and 1:1000 anti-GFP (Santa Cruz Biotechnology) antibodies (4°C overnight); washed in PBS (3 × 10 min at 25°C); and then subjected to reaction with fluorescence-conjugated secondary antibodies of 1:1000 Alexa Fluor 488 and 1:1000 Alexa Fluor 568 ; and rinsed with PBS (3 × 10 min at 25°C). The sections were mounted on slides using sterile 0.2% gelatin and DAPI mounting media (Vector Laboratories) and coverslipped. Images were taken using either an Axiovert 200M Fluorescent Microscope (Zeiss) equipped with a 20× objective or an LSM 710 Confocal Microscope (Zeiss) equipped with a 63× objective. For the immunohistochemistry, the following two sets of control experiments were performed to test specificity: (1) replacement of the primary antibody with only the serum of the appropriate species; and (2) omission of secondary antibodies. No immunostaining was detected under either of these conditions.Serial sections (30 µm) of NAc  and CeA (AP 0.00 to −2.00 mm) were mounted on slides with DAPI mounting media (Vector Laboratories) and visualized using an Axiovert 200M Fluorescent Microscope (Zeiss) equipped with a 10× objective to assess the location of the injection site. The quality of injection was quantitatively scored based of the strength of EGFP viral expression, injection location relative to target, and the spread of the virus. The injection was considered to be on target if the needle placement was within 0.3 mm of the desired stereotaxic coordinates and the virus EGFP expression covered at least one-third of the brain region of interest  on at least one side of the brain.http://imagej.nih.gov/ij/). Composite images were split into individual channels and overlaid with a grid, and colocalized cells were counted. An LSM 710 Confocal Microscope (Zeiss) equipped with a 63× objective was used to take representative images for IKKβ cell-type specificity viral trophism.Brain sections were prepared as described in the Immunohistochemistry subsection. Epifluorescent images were acquired using an Axiovert 200M Fluorescent Microscope (Zeiss) equipped with a 20× objective and an automated stage. Images of the brain region of interest were captured  then were stitched together creating a composite view for further analysis. Images were taken without saturating the signal and digitized at 8 bits using the full intensity range of 0–256, and imported into the ImageJ software package . Calculated p values <0.05 were considered to be statistically significant.Numerical data are shown as the mean ± SEM, and F = 6.9, p < 0.05) and preference  = 8.3, p < 0.01) 6 h after administration  = 24.1, p < 0.001) and preference  = 12.4, p < 0.01; We first investigated the effects of systemic IKKβ inhibition on voluntary ethanol drinking. A pharmacological approach was selected because IKKβ genetic deletion causes embryonic lethality due to liver degeneration and apoptosis . Low andstration A,B. BothF = 14.0, p < 0.01) and preference  = 21.6, p < 0.01) without affecting total fluid intake  = 31.7, p < 0.001; We administered TPCA-1 (50 mg/kg) or sulfasalazine (100 mg/kg) daily to a different cohort of adult male C57BL/6J mice and performed a 2BC test with limited 3 h access to 15% ethanol during the dark phase of the light/dark cycle, referred to as the 2BC-DID test. Compared with the continuous 2BC test, the 2BC-DID paradigm more closely replicates binge drinking, where mice typically consume higher levels of ethanol and exhibit behavioral evidence of intoxication . In thisd intake A–C. Sulf. The CeA was selected because it is in involved in activating brain stress systems through the release of corticotropin-releasing factor and it negatively reinforces addictive behaviors (Ikkb deletion (F/FIkkb) were injected bilaterally in the brain region of interest with a lentivirus expressing either Cre fused to EGFP (LV-EGFP-Cre) or only EGFP (LV-EGFP-Empty). The transgenes of both viral vectors were under the control of a CMV promoter and were pseudotyped with VSV-G. Expression of Cre results in the excision of Ikkb. This method of targeted IKKβ deletion was validated by injecting LV-EGFP-Cre (n = 8) and LV-EGFP-Empty (n = 8) in the NAc of adult male F/FIkkb mice followed by a 3- or 8-week incubation period. The time points were selected based on previous work in mouse brain showing maximal changes in expression 2 to 4 weeks post-injection (p < 0.01) at 3 weeks and 0.099 ± 0.023 (p < 0.001) at 8 weeks . The NAc was chosen because it is part of the mesolimbic dopamine reward system that positively reinforces addictive behavior . The NAc. The CeAehaviors . Mice genjection . In addi 8 weeks . These rF/FIkkb mice were injected bilaterally with LV-EGFP-Cre or LV-EGFP-Empty into either the NAc or CeA. After 4 weeks, the 2BC drinking test, in which mice could drink either water or a series of increasing ethanol concentrations ranging from 3% to 16%, was administered. Similar to the results after peripheral inhibition of IKKβ, targeted deletion of IKKβ in the NAc also reduced ethanol consumption  = 10.0, p < 0.005) and preference  = 8.3, p < 0.01) without affecting total fluid intake  = 19.1, p < 0.0001) and preference  = 23.9, p < 0.0001) with no change in total fluid intake  = 69.97, p < 0.0001) and CeA  = 53.43, p < 0.0001), but no effect of treatment  or treatment × concentration interaction . Analysis of total fluid intake revealed no significant differences between the LV-EGFP-Cre and LV-EGFP treatment groups . Thus, the knockdown of IKKβ in either the NAc or CeA did not change the preference for saccharin.Because ethanol drinking behavior in the 2BC test depends partly on taste , we inveIkkb and downstream proinflammatory cytokines in the NF-κB canonical pathway. To assess needle placement and viral spread, animals were perfused and brains harvested from a subset of the lentiviral-treated F/FIkkb mice used in the brain region-specific IKKβ knock-down experiments . Injection coordinates and coverage of the NAc and CeA were verified using immunofluorescence to detect EGFP. F/FIkkb mice treated with either LV-EGFP-Cre or LV-EGFP-Empty. The left side of the fluorescent image shows the EGFP signal  in green and DAPI  in blue. The right side of the image is a bright-field image used to better visualize the neuroanatomical landmarks. Upon completion of the behavioral experiments (∼8 weeks post-injection), the knockdown of IKKβ in the NAc and CeA was verified by (1) anatomical assessment of needle placement and viral spread, (2) confirmation of IKKβ protein knockdown, and (3) exploration of changes in mRNA levels of n = 5, LV-EGFP-Cre; n = 5, LV-EGFP-Empty). Brains were prepared, immunostained, and analyzed in the same manner as the IKKβ knock-down experiment (after 3 and 8 weeks) previously described. The relative expression of IKKβ in Cre-treated animals versus control was 0.122 ± 0.026 (p < 0.001) in the NAc and 0.141 ± 0.028 (p < 0.001) in the CeA  and CeA  experiments were harvested, flash frozen, sectioned, and micropunches were collected at the injection site. The relative expression of Ikkb was 0.321 ± 0.049 (p < 0.001) in the NAc and 0.360 ± 0.056 (p < 0.001) in the CeA; Tnf expression was 0.568 ± 0.059 (p < 0.01) in the NAc and 0.488 ± 0.084 (p < 0.01) in the CeA; and Il6 expression was 0.595 ± 0.055 (p < 0.01) in the NAc and 0.641 ± 0.060 (p < 0.01) in the CeA (mean ± SEM). These values indicate ∼68% and 64% decrease in Ikkb mRNA in the NAc and CeA, respectively, and ≥35% knockdown of Tnf and Il6 mRNA in both brain regions  from three adult male alcohol-naive C57BL/6J mice. Using fluorescent light microcopy to visualize IKKβ signal colocalization, we observed that in both the NAc and CeA, IKKβ was expressed in all three cell types to some degree, but was primarily expressed in neurons .n = 2, NAc LV-EGFP-Cre; n = 2, CeA LV-EGFP-Cre) using an antibody to target EGFP and the same three cell-specific markers described above. EGFP under the control of a CMV promoter in the VSV-G pseudotyped lentiviral vectors was expressed primarily in neurons (74.6 ± 1.3%), slightly in astrocytes (10.8 ± 2.2%), and only marginally in microglia  was used to curate and identify potential gene targets in mice and humans. As shown in We previously examined chronic ethanol-induced changes in gene expression patterns in mouse brain  and usedIKKβ is a critical component in the regulation of the NF-κB inflammatory cascade, but its role in alcohol drinking had not been investigated prior to this study.Inhibiting IKKβ, either peripherally or in brain regions associated with addictive behaviors, decreased voluntary ethanol consumption and preference in several drinking tests, including long-term and binge-like paradigms. Systemic administration of the peripherally acting IKKβ inhibitors, TPCA-1 or sulfasalazine, reduced ethanol drinking in two distinct drinking models (2BC and 2BC-DID). The ability of sulfasalazine and TPCA-1 to penetrate the BBB is not well established, and their anti-inflammatory effects are thought to be confined to the periphery . HoweverKnockdown of IKKβ in the NAc or CeA was sufficient to decrease voluntary 2BC ethanol consumption, showing that drinking behavior can be selectively regulated by the central actions of IKKβ. The NAc is part of the mesolimbic dopamine reward system, which has a well documented role in substance abuse, and has also been implicated in the rewarding effects of cocaine mediated by IKKβ . The CeANFKB1 gene has also been linked with alcoholism  in both regions. Although the corresponding reductions in these inflammatory cytokines suggest that this pathway is responsible for the decreased drinking, these results do not provide specific mechanistic evidence of downstream effects. Other studies  have hypcoholism .In addition, previous evidence suggests that long-term ethanol consumption alters gene expression of IKKβ in mouse PFC  and humaCre favored the transduction in neurons, and to a lesser degree in astrocytes, and only marginally in microglia. Even though IKKβ was knocked down to some extent in all three cell types, neurons appear to be a primary target. Because GFAP only labels a subset of astrocytes in the CNS and is not an ideal marker, our estimation that IKKβ knockdown occurred in only 10% of astrocytes may be an underestimation. While this caveat warrants the consideration of the role of IKKβ in astrocytes, it does not detract from the novel evidence that neurons are involved in the IKKβ-mediated reduction in ethanol drinking.In addition to different brain regions, different cell types may play unique roles in inflammatory responses . InflammThe IKK complex  is a crucial mediator for several proinflammatory pathways that ultimately result in the activation of NF-κB. IKKβ primarily regulates the NF-κB canonical pathway (transcription of inflammatory genes/antiapoptosis), IKKα regulates the NF-κB noncanonical pathway (cell cycle regulation/proliferation), while IKKγ/NEMO participates in both pathways . We suggThe central effects of IKKβ are not well known, and prior to this work its role in alcohol drinking had not been investigated. Our results provide novel evidence that peripheral and/or central inhibition of IKKβ decreases ethanol drinking, including binge-like consumption. Ethanol could induce peripheral cytokines that ultimately activate expression of immune-related genes in the brain or could directly stimulate central immune- and inflammatory-related pathways. Inhibiting IKKβ-mediated signaling could dampen the peripheral as well as the central inflammatory effects of ethanol. Our results are consistent with other studies showing that null mutant mice lacking genes associated with proinflammatory pathways had reduced levels of chemokines and cytokines, and reduced voluntary ethanol consumption . HoweverIn summary, voluntary ethanol drinking was decreased by inhibiting IKKβ peripherally using pharmacological inhibitors or centrally using genetic deletions in the CeA or NAc, regions known to be important in the neurobiology of alcohol abuse . Althoug


Ctnnb1 gene is associated with different liver diseases including cancer and cholestasis.Wnt/β-catenin signaling plays a crucial role in embryogenesis, tissue homeostasis, metabolism and malignant transformation of different organs including the liver. Continuous β-catenin signaling due to somatic mutations in exon 3 of the Ctnnb1CAhep mice were characterized by reduced body weight, significantly enlarged livers with hepatocellular fat accumulation around central veins and increased hepatic triglyceride content. Proteomics analysis using whole liver tissue revealed significant deregulation of proteins involved in fat, glucose and mitochondrial energy metabolism, which was also reflected in morphological anomalies of hepatocellular mitochondria. Key enzymes involved in transport and synthesis of fatty acids and cholesterol were significantly deregulated in livers of Ctnnb1CAhep mice. Furthermore, carbohydrate metabolism was substantially disturbed in mutant mice.Expression of a degradation resistant form of β-catenin in hepatocytes resulted in 100% mortality within 31 days after birth. Continuous β-catenin signaling in hepatocytes results in premature death due to severe disturbances of liver associated metabolic pathways and mitochondrial dysfunction.Ctnnb1CAhep) and their WT littermates by serum biochemistry, histology, electron microscopy, mRNA profiling and proteomic analysis of the liver.To investigate the influence of permanent β-catenin signaling on liver biology we analyzed mice with hepatocyte specific expression of a dominant stable form of β-catenin ( The role of β-catenin has been extensively investigated, highlighting its importance for organogenesis, pattern formation, cell proliferation, tissue regeneration and apoptosis in different organs including the liver. Along these lines it has been shown that β-catenin is temporally active during liver development and is essential for hepatic morphogenesis and metabolic zonation , 2. In aCtnnb1hepΔ) results in underdeveloped livers characterized by immature hepatocytes, canalicular abnormalities, bile secretion defects and hence cholestasis and fibrosis -FDG -FDG . TherefoIn summary, our study provides further evidence linking β-catenin signaling to metabolic deregulation and down-regulation of genes involved in energy-producing processes as observed in a subgroup of liver cancer patients, especially in the pediatric subtype of liver cancer.However, further studies are necessary to understand the impact and complexity of continuous β-catenin signaling in hepatocytes.Ctnnb1CA hep and Ctnnb1TCCAhep mice were generated as described before [Ctnnb1CA hep mice were sacrificed at the age of 21 days. Ctnnb1TCCAhep mice were injected with tamoxifen at the age of 10 weeks and sacrificed 12 days afterwards.d before . All CtnCre-negative littermates were exclusively used as controls throughout the entire study. All experiments performed in this study were approved by the local ethical committee (BMWFW-66.009/0134-WF/V/3b/2015).Serum parameters were quantified using standard methods as described previously .Liver tissue was processed as described previously . Toluidi® Reagent RNA Isolation Reagent (Sigma Aldrich) from snap frozen liver tissue following the instructions of the manufacturer. Complementary DNA (cDNA) was generated from 2 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the instructions of the manufacturer. iQ™ SYBR® Green Supermix (BioRad) was used for quantitative PCR (qPCR). Primer sequences were obtained from the qPrimerDepot (http://mouseprimerdepot.nci.nih.gov) and will be provided upon request. Melting curve analysis and agarose gel electrophoresis was performed to assess the quality of primers and the qPCR.Total cellular RNA was isolated using TRICtnnb1CA hep mice and 3 WT littermates were harvested at the age of 20 days and livers were snap frozen in liquid nitrogen. 2 mg of tissue were homogenized with an ultrasonic homogenizer in Sample Buffer . Then the solution was digested with trypsin.For whole liver proteomic analysis 3 T-test (t2 test to consider the variance between groups (difference of mean) but also the variance within groups. Values of p < 0.05 and 0.58 log2 fold change were considered significant.Proteins were detected by LC (UltiMateTM 3000 RSLCnano) - MS/MS (QExactive Orbitrap) as described before . Obtaine® Pathway Analysis .Proteomic data was analyzed using Ingenuity


Pulmonary fibrosis (PF), characterized by the destruction of lung tissue architecture and the abnormal deposition of extracellular matrix (ECM) proteins, currently has no satisfactory treatment. In the present study, we investigated the hypothesis that XIST play a promotive role in bleomycin (BLM)-induced ECM and pulmonary fibrosis; XIST exerts its effect through miR-139 regulation. XIST expression was upregulated in lung tissues derived from BLM-induced mouse model of PF, and was positively correlated with β-catenin and ECM protein levels, respectively. LV-sh-XIST-induced XIST knockdown led to decreased PF, reduced β-catenin and ECM protein levels in lung tissues. XIST knockdown suppressed the proliferation of IMR-90 (human fibroblast) and murine lung fibroblasts (MLFCs) and ECM protein expression. Moreover, miR-139 could directly bind to XIST and the 3’UTR of β-catenin; XIST competed with β-catenin for miR-139 binding both in IMR-90 and MLFCs. In MLFCs, miR-139 inversely regulated XIST, and could partially reverse the effect of XIST on β-catenin and ECM proteins. In lung tissues of PF mice, miR-139 expression was downregulated, whereas β-catenin expression was upregulated. In conclusion, XIST exerts positive effects on BLM-induced PF through inhibiting miR-139 to promote human/mouse fibroblast proliferation and ECM proteins. Pulmonary fibrosis (PF) is a devastating chronic lung condition with poor prognosis. Pulmonary fibrosis progresses insidiously and slowly, with acute exacerbation of interstitial pneumonia being a highly lethal clinical event. Furthermore, the mortality rate for pulmonary fibrosis is increasing , 2.It has been reported that pulmonary fibrosis is induced by repeated epithelial cell damage and abnormal wound repair and remodeling, resulting in over-proliferation of fibroblasts and abnormal deposition of extracellular matrix (ECM) proteins, such as collagen I and α-SMA . An incrRecently, a group of long non-coding RNAs , which are non-protein coding transcripts longer than 200 nucleotides , have beThe mechanisms by which lncRNAs exert their effect varies under different conditions, however, emerging evidences have revealed that the interaction between lncRNAs and microRNAs plays a major role , 13. A pAccording to previous studies, several signaling pathways were involved in fibrosis progression; among them Wnt/β-catenin has been reported to play an essential role . RecentlIn the present study, we constructed BLM-induced mouse model of PF, evaluated the detailed functions of XIST in PF mouse, pulmonary fibroblasts proliferation and ECM protein expression in IMR-90 and murine lung fibroblasts (MLFCs). By using online tools, we found that XIST and β-catenin share an almost identical binding site of miR-139. Through Wnt/β-catenin signaling, XIST/miR-139 regulates ECM protein expression in MLFCs. Taken together, we revealed the promotive effect of XIST on BLM-induced PF, and demonstrated the mechanism by which XIST/miR-139 axis exerts its effect on regulating pulmonary fibrosis, providing a potential therapy to treat PF.BLM-induced mouse model of PF was established according to previous study . A totalSince PF is commonly considered the result of a recurrent injury to the alveolar epithelium followed by an uncontrolled proliferation of fibroblasts , next weWe have revealed that XIST knockdown reduced β-catenin and ECM protein levels in PF mice tissues; next we determined β-catenin and ECM protein levels in IMR-90 and MLFCs. β-catenin protein levels in nucleus (compared to Histone H3) and cytoplasm (compared to β-actin) of IMR-90 and MLFCs were determined by using Western blot assays; results showed that after XIST knockdown, β-catenin protein levels were significantly reduced in either nucleus (compared to Histone H3) or cytoplasm (compared to β-actin) of IMR-90 and MLFCs Figure . ConsistLncRNAs can serve as a ‘sponge’ to microRNAs and prevent them from binding to mRNAs based on the competing endogenous RNA (ceRNA) hypothesis . We founWe have revealed that XIST competes with β-catenin for miR-139 binding; next we further investigated the functional role of miR-139 in XIST regulating β-catenin and ECM proteins in MLFCs. After sh-XIST-induced XIST knockdown, miR-139 expression was upregulated Figure . miR-139MiR-139 and β-catenin expression in PF and normal tissues were determined by using real-time PCR assays. Results showed that in PF tissues, miR-139 was downregulated, whereas β-catenin was upregulated Figure  and 6B. Recently, more and more studies demonstrated that PF could be regulated by lncRNAs . Tang etLncRNAs may regulate gene expression in many ways during pulmonary fibrosis. New studies showed that lncRNAs act as key ceRNAs , 23, 24,After co-introducing sh-XIST and miR-139 inhibitor into MLFCs, β-catenin, Collagen I and α-SMA protein levels were monitored. In response to XIST knockdown, β-catenin in nucleus and cytoplasm, Collagen I and α-SMA protein levels were reduced; in response to miR-139 inhibition, the indicated protein levels were increased. Moreover, miR-139 inhibition partially reversed the effect of XIST knockdown on the indicated protein levels. These all indicated that miR-139 might play a role in PF; XIST possibly exerts its function in promoting PF through competing with β-catenin for miR-139 binding.Further, miR-139 expression was downregulated, whereas β-catenin expression was upregulated in PF tissues. Moreover, miR-139 expression was inversely correlated with XIST and β-catenin, respectively; XIST and β-catenin was positively correlated, further indicating the positive role of XIST in PF, and that XIST competes with β-catenin for miR-139 binding to promote PF progression.In conclusion, we revealed the promotive effect of XIST on BLM-induced PF, and demonstrated the mechanism by which XIST exerts its effect on promoting human and mouse fibroblast proliferation, ECM protein expression and pulmonary fibrosis. The present study provided a potential therapy to treat PF. However, the mechanisms by which XIST exerts its functions vary under different circumstances; other promising targets in PF treatment need further investigation.2 at 37°C.Human pulmonary fibroblast, IMR-90  and mouse pulmonary fibroblast, NIH3T3  were cultured in DMEM medium  supplemented with 10% fetal calf serum , 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified incubator with 5% COAll animal procedures were conducted in accordance with humane animal care standards approved by the Hunan Cancer Hospital Medicine Ethics Committee . All experiments were performed in accordance with relevant guidelines and regulations. Male C57BL/6 mice  were obtained from SLAC China  and maintained under specific pathogen-free conditions. The animals were acclimated to the environment for 1 week prior to treatment.n = 12 for each time point). The lung tissues were collected for RNA extraction, HE staining or Masson’s trichrome assay.The mice were administered with BLM  intratracheally at a dose of 1.5 U/kg dissolved in a total of 0.05 ml of sterile saline. The control groups were treated with 0.05 ml of sterile saline using the same method. The mice were sacrificed on day 28 or 32 (™ Lentivirus Concentration Kit (GeneCopoeia), the viral titers of the lentivirus were determined before infection. HCNSLCs cells were plated at a concentration of 2 × 106 cells/ml, incubated for 16 h. For infection, 1.5 ml/well viral supernatant was used to replace the medium. Cells were incubated at 37°C for 10 h, and then the fresh media was used to replace the viral supernatant. 48 h after the infection, 2 mg/ml puromycin was used to select cells. 5 days later, the infection efficiency was verified using Western blot or real-time PCR assays.To generate XIST knocked-out or control lentivirus, the pLVX vector containing shRNA XIST or the control sequence were co-transfected into 293 cells together with the plasmids pHelper1.0 and pHelper2.0 (Genechem) that contain the necessary elements for virus packaging. The transfection was generated using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Supernatants containing the lentivirus were harvested at 72 h, and then concentrated with Lenti-Pac28 days after intratracheal treatment of BLM, lentivirus shRNA XIST or the control sequence were dissolved in saline . The lentivirus vectors were freshly prepared for the experiments. The lentivirus with shRNA XIST or the control sequence was then injected into PF mice tail. 4 more days later, 12 mice were sacrificed and the lung tissues were collected for RNA extraction, HE staining or Masson’s trichrome assay.Lung sections were stained with H&E and Masson’s trichrome and a quantifying assayication of Masson’s trichrome, respectively. Briefly, the severity of fibrosis was graded and scored on a scale of 0–8. Ten fields per section at ×200 magnifications were randomly selected per mouse, and two blinded pathologists carefully and independently examined 60 fields per group using Nikon DS-Fi1-U2 microscope .™ 2000 transfection reagent , followed by the protocol recommended by the manufacturer. After 48 h transfection, the cells were collected and used for further investigations.Both the miRNA mimics and miRNA inhibitors were synthesized by Genepharma Company . Oligonucleotide and plasmid transfection were conducted by using the Lipofectamine−ΔΔCT method was used to analyze the relative fold changes.TRIZOL Reagent  was used to extract total RNA. Then RNA sample reverse transcription was achieved by High Capacity cDNA Reverse Transcription Kit . The Fast Start Universal SYBR Green Master  was applied for the quantitative RT-PCR. 2Cell lysates were lysed by RIPA buffer  with Complete Protease Inhibitor Cocktail . Cell lysates were transferred to 1.5 mL tube and kept at −20°C before use. SDS-PAGE was conducted to separate the cellular proteins. And all the cellular proteins within this study were separated by 5% stacking gel and 10% running gel. The molecular weight of candidate proteins was referred to the information of the Pre-stained SeeBlue rainbow marker  loaded in parallel. The membranes were probed with the following antibodies: β-catenin , Collagen I , α-SMA , Histone H3 , β-actin (Cat# ACTN05 (C4), Abcam) and GAPDH . The blots were detected on Kodak film developer .4/well) were seeded in 96-well plates in triplicate. The cells were serum-starved the following day and infected with 50 nM of either the sh-XIST or the sh-NC controls. The MTT  was added to a final concentration of 0.5 mg/ml, and the cells were incubated for 4 h at 37°C. The absorbance at 490 nm was measured by micro plate reader . Each experiment was repeated at least three times.Cell proliferation was measured by 3--2, 5-diphenyltetrazolium bromide (MTT) assay. IMR-90 and MLFCs (2.5 × 10IMR-90 and MLFCs treated with sh-NC or sh-XIST (10 ng/ ml) were mixed into 0.5 ml of 0.35% agar containing growth medium and layered over a base of 0.5% agar to prevent anchorage-dependent cell growth. Once this layer was solidified, it was overlaid with 1 ml of normal growth medium, which was replaced every 2 days for 14 days. A colony is defined as a cell aggregate larger than 100 mm. Pictures were taken and visible colonies were counted after 14 and 28 days.IMR-90 and MLFCs were seeded into a 24-well plate. After cultured overnight, cells were co-transfected with the wild-type and mutated XIST or wild-type and mutated β-catenin 3′UTR reporter plasmid or pRL-TK plasmids and miR-139 mimics or miR-139 inhibitor. Luciferase assays were performed 48 h after transfection using the Dual Luciferase Reporter Assay System .P < 0.05 was considered statistically significant.The data are presented as mean values, and the error bars indicate standard deviation (SD). The quantitative variables between two groups were compared using the independent-samples t test, and among more groups via one-way analysis of variance (ANOVA) using Dunnett’s test (day 0 as the control group).


PRMT5) in regulating inflammation, cell proliferation, migration and invasion of fibroblast‐like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA). FLSs were separated from synovial tissues (STs) from patients with RA and osteoarthritis (OA). An inhibitor of PRMT5 (EPZ015666) and short interference RNA (siRNA) against PRMT5 were used to inhibit PRMT5 expression. The standard of protein was measured by Western blot or immunofluorescence. The excretion and genetic expression of inflammatory factors were, respectively, estimated by enzyme‐linked immunosorbent assay (ELISA) and real‐time polymerase chain reaction (PCR). Migration and invasion in vitro were detected by Boyden chamber assay. FLSs proliferation was detected by BrdU incorporation. Increased PRMT5 was discovered in STs and FLSs from patients with RA. In RA FLSs, the level of PRMT5 was up‐regulated by stimulation with IL‐1β and TNF‐α. Inhibition of PRMT5 by EPZ015666 and siRNA‐mediated knockdown reduced IL‐6 and IL‐8 production, and proliferation of RA FLSs. In addition, inhibition of PRMT5 decreased in vitro migration and invasion of RA FLSs. Furthermore, EPZ015666 restrained the phosphorylation of IκB kinaseβ and IκBα, as well as nucleus transsituation of p65 as well as AKT in FLSs. PRMT5 regulated the production of inflammatory factors, cell proliferation, migration and invasion of RA FLS, which was mediated by the NF‐κB and AKT pathways. Our data suggested that targeting PRMT5 to prevent synovial inflammation and destruction might be a promising therapy for RA.To probe the role of protein arginine methyltransferase 5 ( Rheumatoid arthritis (RA) is a chronic disease, which is characterized by the hyperplasia of synovial tissues (STs) and progressive destruction of articular cartilage, bone and ligaments Arginine methylation is an abundant post‐translational modification that occurs in mammalian cells and is catalysed by proteins of the arginine methyltransferase (PRMT) family. PRMT5, a member of PRMT family, is localized to both the cytoplasm and the nucleus of mammalian cells 2. After centrifugation, the cells were fostered in DMEM/F12 medium that containing 10% FBS, 100 U/ml penicillin and 100 μg/ml strep at 37°C in a constant humidified incubator of 5% CO2, 21% O2 and 75% N2. The cells were trypsinized when the cells reached assemble and transited. The cells at passages of 3–7 were used in this study. Normal control FLSs were isolated from STs obtained by arthroscopic biopsy from patients that had menisci injuries without history of acute or chronic arthritis, which were kind gifts from Prof. Hanshi Xu in the First Affiliated Hospital of Sun Yat‐Sen University. The study was sanctified by the Medical Ethical Committee of the First Affiliated Hospital, SYSU, China, and executed on the basis of the Declaration of Helsinki.STs were gained from RA patients or osteoarthritis (OA) patients, who went through synovectomy or joint arthroplasty. Briefly, STs were incised into pieces by pieces, then digested with collagenase I  in DMEM/F12 medium  for 3 hrs at 37°C, 5% COSTs were placed in 10% formalin overnight, paraffin‐imbed, incised into 5‐μm sections and put to 3‐aminopropyltriethoxysilane‐coated slides. Then, the sections were deparaffinized by xylene and ethanol which was rehydrated, and using microwave heating with antigen retrieval. Endogenous peroxidase activity was then inhibited by hatching the parts in 1% hydrogen peroxide about 30 min. The segment were then fostered with main antibodies against PRMT5  at 4°C overnight. Then, the samples was washed three times with PBS for 5 min. each and hatched with the respective horseradish peroxidase (HRP)‐conjugated secondary antibodies, substrates and counterstained with haematoxylin.For the transfection of PRMT5 siRNA , FLSs were fostered in six‐well plates; 100 nM siRNA and 10 mg/ml lipofectin in serum‐free medium as a transfection mixture was added to medium‐aspirated cells for 6 hrs. Then, the cells before experiments were incubated with total including 10% FBS DMEM/F12 for 48 hrs.in vitro invasion test was performed by inserting a Matrigel basement membrane matrix . Under a microscope, numbers of invaded cells were randomly selected and counted in 10 high‐power fields. These experiments were performed three times.Chemotaxis analysis of FLSs was preformed through the transwell  migration assay. Filled with DMEM/F12 medium containing 10% FBS, the bottom chambers were used as a chemical enticement. The top chambers were cultured in 200 μl DMEM/F12 medium (without FBS) and FLSs with PRMT5‐specific inhibitor EPZ015666  or FLSs transfected with PRMT5 siRNA for 48 hrs. After 12 hrs, through a cotton swab, the cells which had not migrated were removed from the filter top. The migrated cells, on the bottom of the membrane, were immersed in methanol and dyed with 0.1% Crystal Violet. Through the ZEISS digital microscope dealing with images, we counted the stained cells for each analysis via the mean number of cells per five random areas. The 5 cells/ml, where 90% confluence allowed one parallel wound. The following day, wounds were created in a cell monolayer using 1‐ml sterile micropipette tips. Then starving media were used to wash detached cells, and RA FLSs were treated with or without 10% FBS. After 24 hrs of incubation, migration was quantified with Image J software by counting the cells that moved beyond a reference line.Wounding migration assays were performed as described previously 4 cells/well. Then, RA FLSs were grown to 80% confluence and were pre‐treated with or without EPZ015666 or transfected with siRNA for 48 hrs and then treated with TNF‐α or IL‐β for 24 hrs. The cell multiplication was detected through a Cell‐Light EdU DNA Cell Proliferation Kit . Each test was replicated three times according to the manufacturer's recommendation.5‐Ethynyl‐2′‐deoxyuridine (EdU) is a thymidine analogue; when cells are dividing, it is incorporated into replicating DNA and then applied to sign proliferating cells. RA FLSs were trypsinized, seeded into 96‐well plates and then calculated with a density of 1 × 105 cells/ml. When became approximately 60% confluent, the FLSs were stimulated with IL‐1β or TNF‐α for 24 hrs. Then, they were disposed with paraformaldehyde and infiltrated with PBS containing 0.1% Triton X‐100. The cells were hatched with anti‐PRMT5 antibody all night to measure PRMT5. The cells were then incubated with DAPI, and the coverslips were put on the glass slides with antifade equipment media and then examined by a confocal fluorescence microscopy .RA FLSs or OA FLSs were seeded on sterile cover glass in 35‐mm dishes at a density of 1 × 10After the designated treatments with EPZ015666 or transfected with siRNA for 48 hrs and then treated with TNF‐α or IL‐1β for 12 hrs, total RNAs were extracted using TRIzol (Sigma‐Aldrich) and were reverse‐transcribed to cDNA using miScript Reverse Transcription Kit . The mRNA expression of PRMT5, IL‐6 and IL‐8 was analysed by real‐time quantitative polymerase chain reaction (qPCR) which was performed on cDNA using QuantiTect SYBR Green RT‐PCR Kit on StepOnePlusTM Instantaneous analyse PCR System . The expression of relative mRNA was more normalized to the GAPDH. The used RT‐PCR primers were listed in Table S1.5 cells were seeded. When subconfluence (~80%) was reached, RA FLSs were pre‐processed with EPZ015666 for 24 hrs or transfected with siRNA for 48 hrs. Then, RA FLSs were stimulated with or without TNF‐α for 10 min. Cells were lysed with cell lysis buffer (CST) in an ice bath for 15 min., and then, the lysates were centrifuged at 14000 g for 15 min. at 4°C. Supernatants were incubated with 2× laemmli sample buffer (Sigma‐Aldrich) for 5 min. at 100°C. The samples with same amounts were then separated with SDS‐PAGE gel and shifted to NC membranes and immunoblotted with the indicated antibodies: antiphosphor‐AKT , anti‐AKT , anti‐NF‐κB p65 , antiphosphor‐IKBα , anti‐IKBα  and antiphosphor‐IKK .For each Western blot experiment, 2 × 105 cells/ml, 80% cell confluence) culture supernatants were collected for the measurement of IL‐6 and IL‐8 by enzyme‐linked immunosorbent assay (ELISA), through employing a commercial tool on the basis of the guidance of the manufacturer .After the designated treatments with EPZ015666 or transfected with siRNA for 48 hrs and then disposed with TNF‐α for 12 hrs, RA FLSs'  for 48 hrs. The supernatants were removed. The adherent cells were cultured with 3‐‐2,5‐diphenyltetrazolium salt solution (1 mg/ml in PBS) at 37°C for 30 min. Dark blue formazan was melted in acidified isopropanol, and formazan quantification was performed at a trial wavelength of 570 nm. The wavelength of 620 nm was used as a reference.t‐test or anova. P values less than 0.05 were significant.The software of SPSS 13.0  was utilized. Data were present as means ± standard deviation of the mean (S.E.M.). The differences between each group were detected by We first investigated the expression of PRMT5 in STs. Positive stained cells of anti‐PRMT5 antibody were more prominent in STs from RA when compared with OA patients Fig. A. We nexRA FLSs were stimulated with TNF‐α or IL‐1β. As shown in Figures To investigate the role of PRMT5 on inflammatory response in RA FLSs, a specific PRMT5 inhibitor (EPZ015666) or transfected with PRMT5 siRNA were used. As shown in Figure Recent studies indicated that inhibition of PRMT5 suppressed proliferation in tumour cell lines. Therefore, we explore whether PRMT5 regulates proliferation of RA FLSs, which was measured by EdU incorporation. We showed that pre‐treatment with EPZ015666 and PRMT5 siRNA reduced TNF‐α‐induced proliferation of RA FLSs Fig. C. FurtheMTT test was used to evaluate the toxic effect of EPZ015666 on RA FLS viability. Up to 5 μM, EPZ015666 did not reduce the viability of RA FLSs, which indicated that the inhibitory effects of EPZ015666 were not as a result of drug cytotoxicity (data not shown).Then, we explored the role of PRMT5 in the invasion and migration of RA FLSs using a specific PRMT5 inhibitor (EPZ015666) or PRMT5 siRNA transfection. Treatment with EPZ015666 and PRMT5 siRNA markedly inhibited FBS‐induced migration of RA FLSs Fig. A. Wound Under proinflammatory stimulation, activation of NF‐κB pathway required the activation of IκB kinase (IKK). Activated IKK promoted the phosphorylation and degradation of the inhibitor of NF‐κB (IκB), and phosphorylation of p65. We further explore the association between PRMT5 and NF‐κB. As shown in Figure Because AKT was an important factor in regulating inflammation and migration of RA FLSs, we further investigated the relationship between PRMT5 and AKT. It was observed that EPZ015666 treatment inhibited phosphorylation of AKT in TNF‐α‐treated RA FLSs  residues of the p65 subunit, and consequently regulated NF‐κB‐dependent gene expression, such as IL‐1α and TNF receptor‐associated factor 1. Additionally, it has also been reported that R35 as well as R30 at p65, which was methylated by PRMT5, participated in transcription of TNF‐α‐induced pro‐inflammatory genes in endothelial cells We observed that PRMT5 inhibition suppressed TNF‐α‐induced phosphorylation of IKKβ and IκBα, as well as translocation of nuclear NF‐κB. These data indicated that PRMT5 regulated the NF‐κB pathway by interfering in early cytoplasmic IKK signalling. Similar findings were also indicated in other studies. According to Wei et al. Simulated by TNF‐α, phosphoinositide AKT was crucial in regulating cell proliferation and inflammation in RA In conclusion, we showed that PRMT5 played an important role in inflammatory response, cell proliferation, migration and invasion of RA FLSs through NF‐κB and AKT pathway. Thus, inhibition of PRMT5 might be a potential therapeutic agent to attenuate pathological progression in rheumatoid arthritis.The authors confirm that there are no conflict of interests.Figure S1 PRMT5 inhibition suppressed the inflammatory factors and cell proliferation in IL‐1β RA FLSs.Click here for additional data file.Table S1 The sequences of RT‐PCR primers.Click here for additional data file.


Nuclear PRMT5 expression was inversely correlated with p16-status (p < 0.001) and was significantly higher in tumor samples from patients who smoked > 10 pack-years (p = 0.013). In addition, nuclear PRMT5 was directly correlated with cyclin D1 (p = 0.0101) and IL-6 expression (p < 0.001). In a subgroup survival analysis, nuclear PRMT5-positive/IL-6-positive group had worst survival, whereas nuclear PRMT5-negative/IL-6-negative group had the best survival. Similarly, patients with p16-negative/nuclear PRMT5-positive tumors had worse survival compared to patients with p16-positive/nuclear PRMT5-negative tumors. Our mechanistic results suggest that IL-6 promotes nuclear translocation of PRMT5. Taken together, our results demonstrate for the first time that nuclear PRMT5 expression is associated with poor clinical outcome in OPSCC patients and IL-6 plays a role in the nuclear translocation of PRMT5.Protein arginine methyltransferase-5 (PRMT5) plays an important role in cancer progression by repressing the expression of key tumor suppressor genes via the methylation of transcriptional factors and chromatin-associated proteins. However, very little is known about the expression and biological role of PRMT5 in head and neck cancer. In this study, we examined expression profile of PRMT5 at subcellular levels in oropharyngeal squamous cell carcinoma (OPSCC) and assessed its correlation with disease progression and patient outcome. Our results show that nuclear PRMT5 was associated with poor overall survival ( Head and neck squamous cell carcinoma (HNSCC) is the eighth most frequent cancer worldwide and five-year survival rate < 50%) is among the lowest of the major cancers , 2. Toba0% is amoRecent studies have shown that protein arginine methyltransferases (PRMTs) regulate a number of cellular processes, including proliferation, apoptosis, anoikis and epithelial-mesenchymal transition (EMT), which play an important role in tumor growth and metastasis –18. TherIn this study, we examined the expression and localization of PRMT5 in oropharyngeal squamous cell carcinoma (OPSCC) tumors and correlated it with patient survival, p16 status (an established surrogate for tumor HPV status in OPSCC), smoking status, cyclin D1 expression, IL-6 expression and other clinical and pathological variables. Our results show a direct correlation between nuclear PRMT5 expression and poor overall survival. Nuclear PRMT5 expression was significantly higher in tumor samples from patients that smoked > 10 pack-years and inversely correlated with p16 status. In addition, nuclear PRMT5 expression directly correlated with IL-6 and cyclin D1 expression. In our mechanistic experiments, IL-6 treatment markedly enhanced nuclear translocation of PRMT5 in head and neck cancer cells.To evaluate the expression pattern and clinical importance of PRMT5, cyclin D1 and IL-6 in OPSCC, we assessed the expression of these biomarkers in TMA's constructed using 211 surgically treated samples. Patient characteristics are listed in Table p < 0.012). Patients whose tumors were nuclear PRMT5 positive had a 1.732 hazard ratio of death (95% CI: 1.127–2.661) as compared to those in which PRMT5 was not present in the nucleus . We and others have previously shown that OPSCC patients with HPV-negative tumors have inferior outcomes as compared to patients with HPV-positive patients [p = 0.0043; HR 2.073 95% CI:1.257–3.418). Nuclear PRMT5 expression was inversely associated with p16 expression. A greater proportion of p16-negative tumors expressed higher nuclear PRMT5 as compared to p16-positive tumors . In the subgroup survival analysis, p16-negative/PRMT5 nuclear-positive (p16-/Nuclear PRMT5+) group had the worst survival compared to the other groups .PRMT5 expression was evaluable in 209 tumors and it was predominately cytoplasmic in the OPSCC tumor samples . However, we also observed nuclear PRMT5 expression in 58/209 (27.8%) tumors. Tumors that expressed PRMT5 in the nucleus were categorized as nuclear positive and tumors with cytoplasmic expression were classified as nuclear negative. Higher nuclear PRMT5 expression was associated with poor overall survival . A larger proportion of patients who smoked > 10 pack-years were positive for nuclear PRMT5 expression as compared to patients that smoked ≤ 10 pack-years (24.38% versus 3.98%). In the subgroup survival analysis, PRMT5 nuclear-negative/≤ 10 pack years group had the best survival compared to the other groups .Patients who smoked > 10 pack-year showed poor overall survival and had significantly higher expression of nuclear PRMT5. We and others have previously shown that smokers have poor clinical outcome as compared to non-smokers . In thisp = 0.0009; HR 2.209 95% CI: 1.385–3.523). Patients whose tumors were nuclear PRMT5 positive had higher expression of cyclin D1 .Recent studies have shown that PRMT5 regulates tumor growth by modulating cyclin D1 expression . In addip < 0.001) [p < 0.0001; HR 4.089 95% CI: 2.390–6.995). Interestingly, nuclear PRMT5 expression was directly associated with high IL-6 expression in the primary tumor samples , perineural invasion (p = 0.007), extracapsular spread (p = 0.014) and inversely associated with p16 status (p = 0.011).IL-6 expression was evaluable in 198 tumors and was found to be overexpressed in 112 (56.6%) tumors. Overexpression of IL-6 was significantly associated with worse overall survival of patients with OPSCC (< 0.001) . The hazOur results from this study show that nuclear localization of PRMT5 was directly associated with poor clinical outcome in OPSCC patients. In the same patient cohort, IL-6 overexpression was also associated with poor survival. Interestingly, high expression of IL-6 was directly correlated with nuclear PRMT5 localization, thereby suggesting that IL-6 might be promoting PRMT5 nuclear translocation. To examine the effect of IL-6 on PRMT5 nuclear localization, we performed nuclear/cytosolic fractionation and immunofluorescence staining experiments using head and neck cancer cell lines. Our results show that IL-6 treatment of CAL27 cells markedly enhanced nuclear translocation of PRMT5 Figure . SimilarThe role of PRMT5 in cancer, particularly in regulating the expression of key tumor suppressor genes, has been extensively studied , 15, 18.Over the past decade, it has become apparent that the incidence of “classic” tobacco/alcohol-induced HNSCC has declined, but at the same time, HNSCC caused by HPV has risen sharply . HPV16 iCyclin D1 is a key regulator of cell cycle and is often overexpressed in numerous cancer types including breast, colon, lung and head and neck , 49. OurOropharyngeal squamous cell carcinoma (OPSCC) tissue specimens were obtained from surgical resections of patients at The Ohio State University James Cancer Hospital and Solove Research Institute between 2002 to 2009. All patients underwent surgical resection as a first line of therapy with a curative intent. This was followed by no additional treatment or adjuvant chemotherapy and/or radiotherapy as needed. The Ohio State University Institutional Review Board approved a retrospective analysis study of these specimens and a waiver of HIPAA authorization was obtained. Patient characteristics, including age, race, gender, marital status, smoking status, pathological variables including tumor size, nodal status, AJCC stage, extracapsular spread, perineural invasion and clinical variables including survival and recurrence outcomes were recorded.Paraffin-embedded archival tissue blocks and their matching H&E-stained slides were retrieved from the Department of Pathology. A pathologist marked the areas with cancer and adjacent normal on the H&E slides. Representative regions  were sampled using a 0.6-mm punch on a master TMA blocks. The TMA's were constructed by the Histology Core in the Department of Pathology. Unstained sections were cut and used for immunohistochemical staining.TMA slides were stained to assess the tumor expression of PRMT5, p16, cyclin D1 and IL-6, using immunohistochemistry as previously described . BrieflyCAL27 cells were obtained from ATCC . UM-SCC-74B cell line was obtained from the laboratory of Dr. Thomas E. Carey at the University of Michigan. The idenCytoplasmic and nuclear fractions from tumor cells were separated using the NE-PER Nuclear & Cytoplasmic extraction kit . After IL-6 treatment, cells were harvested and washed with PBS. Cell pellet was treated with cell membrane lysis reagents from the extraction kit to release the cytoplasmic contents. The cytoplasmic proteins were collected by centrifugation leaving the intact nuclei in the pellet. The nuclear pellet was washed with PBS to reduce carryover of the cytoplasmic proteins to the nuclear protein fraction. Nuclear lysis buffer was then added to the pellet to lyse the nuclei and release the nuclear proteins. Reducing agent and loading buffer were then added to the cytoplasmic and nuclear extracts and analyzed by Western blotting.Whole cell lysates or cytosolic/nuclear protein fractions were separated by 4–12% NuPAGE Bis-Tris gels  and transferred onto PVDF membranes using NuPAGE transfer buffer (Invitrogen). To block nonspecific binding, membranes were incubated with 5% Milk in Tris buffered saline containing 0.1% Tween-20 (TBST) or 3% BSA in TBST for 1 hour at room temperature. The blots were then incubated with the respective primary antibody in TBST + 5% Milk or 3% BSA according to manufacturer's instructions at 4°C overnight. After washing with TBST, the blots were incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG  or with donkey anti-rabbit IgG  for 1 hour at room temperature. An ECL-plus Western blotting substrate  was used to detect specific protein bands.Tumor cells were cultured in Labtech chambers in serum free medium for 2 hours and then treated with recombinant IL-6 (50 ng/ml) for 30 minutes. At the end of incubation, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized by treating with 100% methanol for 10 minutes at –20°C. Next, slides were washed with PBS, blocked with normal goat IgG for 1 hour and incubated overnight at 4°C with mouse anti-PRMT5 antibody. After washing with PBS, chamber slides were incubated with secondary antibodies (goat anti-mouse-IgG-Alexa Fluor 488). Chamber slides were then mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). The fluorescent images were captured using Nikon Eclipse 80i microscope with DS-Ri1 camera at 600X magnification and overlaid using NIS-Elements-Basic Research software .Overall survival was defined as time from the date of surgery to date of death, with patients alive at the date of last observation censored. Cox proportional hazards models were used to assess univariate associations of biomarkers or patient characteristics as predictors for death. Unadjusted hazard ratios (HR) and 95% confidence intervals (CI) are reported. Multivariable models were built to estimate adjusted HRs which let us asses marker effects  beyond the effects of T stage, N stage, AJCC stage, smoking status and gender. To assess dual marker interactions, comparisons of survival curves were evaluated using the log-rank test with adjustments for multiple comparisons made by Bonferonni corrections. Mann-Whitney tests were used to assess associations between biomarkers  or demographics (smoking status) and nuclear PRMT5 or Cyclin D1 expression. All analyses were conducted in SAS, version 9.3 .


Peritoneal dialysis (PD)-associated peritoneal fibrosis is a chronic progress which induces ultrafiltration failure. It remains a challenge to prevent the progression of PD-associated fibrosis in clinic practice. Wnt/β-catenin pathway plays important role in many severe fibrotic diseases, here we investigated its contribution to the development of peritoneal damage. We isolated mesothelial cells (MC) from the effluent of PD patients and found that the expressions of Wnt1, Wnt5a, β-catenin, and LEF1 were increased in patients with more than 1-year PD compared with patients who just started with PD (<1 month). The elevated expressions of Wnts and β-catenin were accompanied with changes in the expressions of E-cadherin, α-SMA, COL-I, and FN mRNA and proteins, which are known related to mesothelial-mesenchymal transition (MMT). In addition, treatment with high glucose significantly increased the expression of Wnt1, Wnt5a, β-catenin, and LEF1 as well as the expression of α-SMA, COL-I, and FN in human peritoneal mesothelial cells (HPMC), whereas the expression of E-cadherin was reduced. Dickkopf-1 (DKK-1) is an endogenous inhibitor of Wnt/β-catenin signaling. Overexpression of DKK1 transgene significantly decreased the expression of β-catenin and attenuated the process of MMT as indicated by the decreased expression of α-SMA, COL-I, and FN and the increased expression of E-cadherin. Furthermore, TGF-β1 treatment significantly activated the Wnt/β-catenin pathway in HPMCs, while DKK1 blocked the TGF-β1-induced Wnt signaling activation and significantly inhibited the process of MMT. These data suggest that the canonical Wnt/β-catenin pathway plays an important role in the MMT and fibrosis induced by PD. Peritoneal dialysis (PD) is an alternative treatment to hemodialysis and commonly used for end-stage renal disease. During PD, the peritoneal membrane (PM), composed of a continuous monolayer mesothelial cells (MC), acts as a permeable barrier in the progress of ultrafiltration and diffusion. In this process, long-term exposure to bioincompatible PD solution containing high concentration of glucose and glucose degradation products (GDP) could impair the PM, resulting in the denudation of the MCs, submesothelial fibrosis and angiogenesis, and finally causes the ultrafiltration failure and discontinuation of PD  exposed to HG and TGF-β1, and its possible relationship with MMT changes.2 at 37°C after three time wash. On the next day, non-adherent cells were removed with fresh culture medium added. After the culture of two passages (about 15 days), the collected HPMCs were used for various experiments. The purity of effluents-derived MCs was determined by flow cytometry. The study was approved by the Institutional Review Board and Ethics Committee of The Second Xiangya Hospital, Central South University, China. All participants thoroughly read and then signed the informed consent form.Twenty four patients undergoing continuous ambulatory PD (CAPD) were enrolled, including 12 patients receiving CAPD for more than 1 year, and the other 12 patients receiving CAPD for less than 1 month. Before the first episode of peritonitis occurred, HPMCs were harvested from the effluents of PD patients as described , and cultured with the DMEM/F12 medium (Invitrogen) containing 10% fetal bovine serum . After reaching 80% confluence, the cells were divided as different groups with different culture media and used the serum-free medium for culture. In the experimental group, DMEM/F12 medium was supplemented with D-glucose  at various concentrations  or with D-mannitol (90 mM), or with recombinant TGF-β1  at gradient  was used for reverse transcription and the cDNA was synthesized as described previously , anti-α-SMA (1:100) and COL-I (1:100) from Santa Cruz Biotechnology; anti-β-catenin (1:100) from Sigma. The antibodies were diluted in 5% BSA and cells were incubated with them overnight at 4°C, followed by incubation with FITC-labeled secondary antibody (Santa Cruz Biotechnology) at 22°C for 2 h. Nuclei was counterstained with 4,6-diamidino-2-phenylindole (DAPI). A UV fluorescence microscope  was used to visualize the immunostained cells and the intensity was analyzed with the image analysis software .pcDNA3.0-FLAG-DKK1 was a generous present from Prof. Youhua Liu . HMrSV5 cells were seeded in 6-well plates previously. After 12 h, the pcDNA3.0-FLAG-DKK1 or pcDNA3.0 empty vector as control was mixed with Lipofectamine  in serum-free Opti-MEMI at room temperature for 30 min according to the manufacturer's instructions and then the mixture was incubated with the HMrSV5 cells at a humidified 5% (v/v) CO2 atmosphere at 37°C for 5 h. To reduce the toxicity of Lipofectamine, cells were washed with fresh complete medium after transfection.t-test was applied for the two group comparison. Normality test has been done before doing ANOVA and t-test. P < 0.05 was considered as statistically significant.The data were presented as mean ± s.e.m. Data were examined using ANOVA (Fisher's protected least significant difference test) for the multiple comparisons. Multiple comparison between the groups was performed using S-N-K method. Two-tailed Student's +CD45−CD68−) was accounted for nearly 96.3% (99.9 × 96.5%) in the cells isolated and cultivated from the effluent of PD patients in both PD > 1 year and PD start groups. The shape of the cells was different in the two groups. Specifically, the cells isolated from the PD-start group had round like shape, but most of the cells from the PD>1 year patients were spindle-shape  was significantly longer than the PD-start group (0.53 ± 0.26 months). The other clinical characteristics, including gender, age, BMI, and renal failure causes, showed no significant differences. The expressions of proteins related to the Wnts pathway  were detected in HPMCs isolated from PD patients. As shown in Figure e Figure .Real-Time-PCR data showed that the expressions of several Wnts, especially Wnt1 and Wnt5a, were significantly upregulated in the PD > 1 year group compared with the PD-start group Figure . The expWe also found that the expressions of MMT-related genes and proteins were also altered. While the expression of E-cadherin was significantly decreased in the PD > 1 year group in both mRNA and protein levels Figures , 2C5,E2,We performed the experiment to examine the high-glucose effect on the HPMCs. The HPMCs were cultured with different concentrations of glucose for 24 h, including normal glucose group (NG: 5.6 mM of glucose), high glucose groups , and osmotic control group (90 mM of mannitol). Interestingly, HPMCs became longer and had fibroblast-like shape when cultured with the increased glucose concentration Figure . The expThe plasmids expressing DKK1 was transfected into HPMCs and then exposed to 60 mM glucose. The immunofluorescence staining data showed that β-catenin was significantly increased in the nuclear region of HPMCs stimulated with high glucose, whereas the signal intensity was restored in cells transfected with DKK1. The expression of α-SMA had similar trend Figure . On the The HPMCs turned into fibroblast-like form after cultured with TGF-β1 Figure . Real-TiHPMCs treated with transfection of DKK1 or mock-transfection of DKK1 were exposed to TGF-β1 and showed that the expression of β-catenin was significantly increased in the nuclear region in cells with mock transfection, and that the signal intensity was restored in cells transfected with DKK1 Figure . Similarin vitro. Importantly, treatment of HPMCs with the Wnt/β-catenin signaling inhibitor DKK-1 led to the inhibition of High Glucose- or TGF-β1-induced MMT.Our data revealed that the main family members of the Wnt/β-catenin signaling pathway, such as Wnts, β-catenin, and LEF1, were significantly increased in HPMCs isolated from the effluents of long-term PD patients. Those changes positively correlated with MMT and peritoneal fibrosis process. Likewise, the Wnt/β-catenin signaling pathway was activated by High Glucose or TGF-β1 treatment in vivo and in vitro  will inevitably lead to peritoneal fibrosis, resulting in the ultrafiltration failure, and increased morbidity as well as mortality in PD patients (Devuyst et al., The detailed functions of Wnt signaling, reported to be associated with biological development and multiple physiological activities, are still unclear and remain to be further clarified. The most important and canonical one, Wnt/β-catenin pathway, is well understood currently. In embryonic development and organ morphogenesis, the Wnt/β-catenin signaling pathway has played a vital role. So it is reasonable that dysregulation of this pathway in adult may be linked to fibroblast biology and fibrosis (Clevers and Nusse, Recent studies have demonstrated that TGF-β1-induced MMT plays a central role in the pathogenesis of peritoneal fibrosis in PD. However, because TGF-β1 could induce the immune and inflammatory responses, the direct inhibition of the TGF-β1 signaling may cause immune suppression. Other signaling pathways, such as Wnt/β-catenin, may be the alternative candidate to prevent the progression of PD-associated fibrosis. Recently, the inhibition of the Wnt signaling pathway has been reported to regulate the fibrotic responses in non-peritoneal tissues (Lin et al., in vitro and in vivo, and this process was interrupted by Wnt antagonist DKK1, suggesting that Wnt/β-catenin pathway is the downstream of TGF-β1(Wang et al., in vitro and in vivo (Sato, in vitro. These data indicate that Wnt/β-catenin signaling acts in a combinatorial manner with TGF-β signal in the progression of PD-related fibrosis.Interestingly, the crosstalk between TGF-β1 and Wnt pathways has been identified for a long time (Medici et al., vo Sato, . Besidesvo Sato, . Our resIn summary, our data revealed that the activation of Wnt/β-catenin signaling pathway mediated the MMT process and peritoneal fibrosis, and that the suppression of Wnt/β-catenin signaling pathway restrained the development of peritoneal fibrosis. These findings may provide information for developing novel therapeutic strategies associated with Wnt/β-catenin signaling pathway for peritoneal fibrosis in patients undergoing PD.This study was carried out in accordance with the recommendations of Institutional Review Board and Ethics Committee of Second Xiangya Hospital, Central South University with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Review Board and Ethics Committee of The Second Xiangya Hospital, Central South University.YG, FL, and LT conceived and designed the study. RG, YF, RW, and YL performed the experiments. All authors analyzed data. YG and LX wrote the paper. LS, FL, LT, and NL reviewed and edited the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.