scholarly journals Elevated microRNA-21 Is a Brake of Inflammation Involved in the Development of Nasal Polyps

2021 ◽  
Vol 12 ◽  
Author(s):  
Ruowu Liu ◽  
Jintao Du ◽  
Jiao Zhou ◽  
Bing Zhong ◽  
Luo Ba ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Nasal Epithelial Cells ◽  
Protein Levels ◽  
Control Subjects ◽  
Luciferase Reporter System ◽  
Suppressive Cytokine

BackgroundCRSwNP is an inflammatory disease but the mechanism is not yet fully understood. MiR-21, a member of miRNAs, has been reported to play roles in mediating inflammation. However, the expression of miR-21 and its role in patients with CRSwNP remain elusive.MethodsTurbinates from control subjects, uncinate processes from CRSsNP, polyp tissues from CRSwNP, and nasal epithelial cells brushed from nasal mucosa were collected. The expression of miR-21 and cytokines in nasal tissues and epithelial cells were detected by qPCR. The localization of miR-21 was detected by ISH, and its target was identified by bioinformation analysis, qPCR, IHC, WB, and luciferase reporter system. The protein and mRNA of PDCD4 and NF-κB P65 were determined by WB and qPCR after miR-21 transfection in HNEpC. The role of miR-21 on cytokines was analyzed in HNEpC and nasal polyp explants.ResultsMiR-21 was upregulated in CRSwNP relative to control subjects by qPCR, which was determined mainly in nasal epithelial cells of CRSwNP by ISH. Both pro-inflammation cytokines (IL-1β, IL-6, IL-8, IL-25, and TSLP) and a suppressive cytokine (IL-10) were overexpressed in the epithelial cells of CRSwNP. The expression of miR-21 was positively correlated with IL-10 and negatively correlated with IL-6, IL-8, IL-33, and TSLP in the epithelial cells of CRSwNP. As a potential target of miR-21, the expression of PDCD4 was negatively correlated with miR-21 in CRSwNP. In HNEpC, miR-21 could reduce the expression of PDCD4 at both mRNA and protein levels, and bioinformation analysis and luciferase reporter system confirmed PDCD4 as one target of miR-21. Furthermore, miR-21 could decrease the activation of NF-κB and increase IL-10 mRNA. Both SEB and LPS could elevate miR-21, with IL-25, IL-33, TSLP induced by SEB and IL-1β, IL-6, IL-8 induced by LPS, while the miR-21 could regulate the expression of IL-33, TSLP, IL-1β, IL- 6 and IL-8 in vitro and ex vivo. Clinically, miR-21 expression was inversely correlated with the Lund-Mackay CT scores and the Lund-Kennedy scores in CRSwNP.ConclusionMiR-21 could be a prominent negative feedback factor in the inflammation process to attenuate the expression of pro-inflammatory cytokines, thereby playing an anti-inflammation role in CRSwNP.

scholarly journals MiR-218 inhibits malignant phenotypes of glioma by targeting TNC/AKT/AP-1/TGFβ1 feedback signaling loop

2020 ◽  
Author(s):  
Siwen Dang ◽  
Rui Zhang ◽  
Sijia Tian ◽  
Banjun Ruan ◽  
Peng Hou ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Malignant Tumors ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Normal Brain ◽  
Reporter System ◽  
Control Subjects ◽  
Luciferase Reporter System

Abstract Background: Gliomas are the most common and malignant tumors in the brain of humans, and the prognosis of glioma patient is very poor. MicroRNAs (miRNAs) play critical roles in different types of cancer by regulating gene expression at the posttranscriptional levels. Although miR-218 has been reported to be downregulated in gliomas, its role in gliomas still remains largely unknown. Methods: MiR-218 expression in gliomas and normal brain tissues (control subjects) were analyzed using TCGA dataset. The biological roles of miR-218 in glioma cells were determined by a series of in vitro and in vivo studies. The dual-luciferase reporter system was performed to identify potential targets of miR-218. The regulatory effect of miR-218 on TNC/AKT/AP-1/TGFβ1 pathway was evaluated by dual-luciferase reporter system and western blot.Results: We demonstrated miR-218 was significantly downregulated in gliomas compared to control subjects, and exerted a potent tumor suppressor in glioma cells by inhibiting cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice, as well as inducing cell cycle arrest and apoptosis.Mechanistically, miR-218 inhibited malignant phenotypes of glioma cells by binding to the 3’ UTR of its target TNC and subsequently repressing its expression. As a result, it could reduce AKT phosphorylation and subsequently inhibit transcriptional activity of AP-1 by reducing JNK phosphorylation, downregulating the expression of TGFβ1, while TGFβ1 is able to, in turn, activate the TNC/AKT/AP-1 signaling axis.Conclusions: Our data uncover a previously unknown tumor suppressor role of miR-218 in glioma by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop.

scholarly journals MiR-218 inhibits malignant phenotypes of glioma by targeting TNC/AKT/AP-1/TGFβ1 feedback signaling loop

2020 ◽  
Author(s):  
Siwen Dang ◽  
Rui Zhang ◽  
Sijia Tian ◽  
Banjun Ruan ◽  
Peng Hou ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Regulation Of Gene Expression ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Normal Brain ◽  
Reporter System ◽  
Control Subjects ◽  
Luciferase Reporter System

Abstract Background: Gliomas are the most malignant and common tumors in human brains, and the prognosis of glioma patient is very poor. MicroRNAs (miRNAs) play critical roles in different types of cancer by performing posttranscriptional regulation of gene expression. Although miR-218 has been demonstrated decreased in gliomas, its role in gliomas still remains largely unknown. Methods: MiR-218 expression were analyzed in gliomas and normal brain tissues (control subjects) using TCGA dataset. A series of in vitro and in vivo studies was performed to determine the biological roles of miR-218 in glioma cells. Potential targets of miR-218 were identified using dual-luciferase reporter system. Western blot and dual-luciferase reporter system were performed to evaluate the regulatory effect of miR-218 on TNC/AKT/AP-1/TGFβ1 pathway.Results: We demonstrated miR-218 was significantly downregulated in gliomas compared to control subjects, and played potent tumor suppressor roles in glioma cells by inhibited cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice, as well as inducing cell cycle arrest and apoptosis.Mechanistically, miR-218 inhibited malignant phenotypes of glioma cells by binding to the 3’ UTR of its target TNC and subsequently repressing its expression. As a result, it could reduce AKT phosphorylation and subsequently inhibit transcriptional activity of AP-1 by reducing JNK phosphorylation, downregulating the expression of TGFβ1, while TGFβ1 is able to, in turn, activate the TNC/AKT/AP-1 signaling axis.Conclusions: Our data uncover a previously unknown tumor suppressor role of miR-218 by blocking the TNC/AKT/AP-1/TGFβ1 positive feedback loop in glioma.

scholarly journals Targeting of the Nasal Mucosa by Japanese Encephalitis Virus for Non-Vector-Borne Transmission

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Obdulio García-Nicolás ◽  
Roman O. Braun ◽  
Panagiota Milona ◽  
Marta Lewandowska ◽  
Ronald Dijkman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Ex Vivo ◽  
Encephalitis Virus ◽  
Nasal Epithelium ◽  
Virus Shedding ◽  
Nasal Epithelial Cells ◽  
Vector Borne

ABSTRACTThe mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles betweenCulexmosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages asex vivoandin vitromodels, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs.IMPORTANCEJEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages asex vivoandin vitromodels, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV.

scholarly journals Involvement of the Extracellular Matrix Proteins Periostin and Tenascin C in Nasal Polyp Remodeling by Regulating the Expression of MMPs

2020 ◽  
Author(s):  
Kun Du ◽  
Min Wang ◽  
Nan Zhang ◽  
Ping Wang ◽  
Pei Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyp ◽  
Nasal Polyps ◽  
Ex Vivo ◽  
Tissue Remodeling ◽  
Tenascin C ◽  
Protein Levels ◽  
Control Subjects

Abstract Background: Tissue remodeling caused by increased MMPs is involved in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). We previously found higher levels of periostin and tenascin C in CRSwNPs, but whether they are associated with the dysregulation of MMPs is unknown. Therefore, the present study aimed to investigate the regulatory roles of two ECM proteins in the expression of MMPs in nasal polyps.Methods：The concentrations of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, TIMP-1, TIMP-2, TIMP-3, TIMP-4, periostin, and tenascin C in tissue homogenates of 51 patients with chronic rhinosinusitis with and without nasal polyps and 15 control subjects were measured and their correlations were analyzed. Primary human nasal polyp fibroblasts and epithelial cells were stimulated ex vivo with periostin and tenascin C and the gene expression of MMPs and TIMPs was determined by means of real-time PCR.Results: The protein levels of MMP-3, MMP-7, MMP-8, MMP-9, TIMP-1, TIMP-2, periostin, and tenascin C were significantly higher in patients with CRSwNPs than in healthy control subjects. Periostin was positively correlated with MMP-3 and TIMP-2, and tenascin C was positively correlated with MMP-3, MMP-7, MMP-8, MMP-9 and TIMP-2. Periostin stimulated the gene expression of MMP-3, MMP-7, and MMP-9 in fibroblasts and MMP-7 in epithelial cells ex vivo. Tenascin C stimulated the expression of MMP-3, MMP-8, and MMP-9 in epithelial cells, but not in fibroblasts. The expression of TIMPs in fibroblasts and epithelial cells was affected by neither periostin nor tenascin C. Conclusions：Periostin and tenascin C might be involved in the remodeling of nasal polyps by regulating the expression of different MMPs in epithelial cells and fibroblasts. Our findings have the potential to identify key factors of tissue remodeling in CRSwNPs.

scholarly journals Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1

2008 ◽  
Vol 89 (7) ◽  
pp. 1569-1578 ◽  
Author(s):  
Maxime Ratinier ◽  
Steeve Boulant ◽  
Christophe Combet ◽  
Paul Targett-Adams ◽  
John McLauchlan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Infectious Clone ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Subgenomic Replicon ◽  
Luciferase Reporter System ◽  
Transcriptional Slippage

Since the first report of frameshifting in HCV-1, its sequence has been the paradigm for examining the mechanism that directs alternative translation of the hepatitis C virus (HCV) genome. The region encoding the core protein from this strain contains a cluster of 10 adenines at codons 8–11, which is thought to direct programmed ribosomal frameshifting (PRF), but formal evidence for this process has not been established unequivocally. To identify the mechanisms of frameshifting, this study used a bicistronic dual luciferase reporter system in a coupled transcription/translation in vitro assay. This approach revealed +1 as well as –1 frameshifting, whereas point mutations, selectively introduced between codons 8 and 11, demonstrated that PRF did not readily account for frameshifting in strain HCV-1. Sequence analysis of cDNAs derived from RNA transcribed by T7 RNA polymerase in the dual luciferase reporter system, as well as in both a subgenomic replicon and an infectious clone derived from strain JFH1, identified additions and deletions of adenines between codons 8 and 11 due to transcriptional slippage (TS). Moreover, RNA isolated from cells infected with virus generated by JFH1 containing the A-rich tract also contained heterogeneity in the adenine sequence, strongly suggesting TS by the NS5B viral polymerase. These findings have important implications for insight into frameshifting events in HCV-1 and demonstrate for the first time the involvement of transcriptional slippage in this recoding event.

The Expression and Regulation of Na+-K+-ATPase in Nasal Epithelial Cells of Chronic Rhinosinusitis with Nasal Polyps

ORL ◽  
2021 ◽  
pp. 1-8
Author(s):  
Guangyi Ba ◽  
Ru Tang ◽  
Song Mao ◽  
Zhipeng Li ◽  
Haibo Ye ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chronic Rhinosinusitis ◽  
Nasal Polyps ◽  
Cell Permeability ◽  
Mrna Levels ◽  
Nasal Epithelial Cells ◽  
Protein Levels ◽  
Control Subjects ◽  
Human Nasal Epithelial Cells ◽  
Ifn Γ

<b><i>Objective:</i></b> Na<sup>+</sup>-K<sup>+</sup>-ATPase (NKA) is essential in maintaining cell permeability, reserving potential energy, and preventing cellular edema. Nevertheless, how NKA expression is altered and regulated in chronic rhinosinusitis with nasal polyps (CRSwNPs) remain uncertain. Therefore, the present study aimed to explore the expression and regulation of NKA in CRSwNP. <b><i>Methods:</i></b> NKA immunolabeling was assessed by the immunohistochemistry method, NKA protein levels were detected with the Western blotting method, and mRNA levels of NKA and aquaporin-5 (AQP5) were assayed by real-time PCR in nasal tissues from CRSwNP and control subjects. The co-localization of NKA with inflammatory cells was evaluated by immunofluorescence staining. In addition, human nasal epithelial cells (HNECs) were cultured and stimulated using various stimulators to evaluate the regulation of NKA. <b><i>Results:</i></b> We found significantly decreased NKA positive cells, NKA protein levels, and mRNA levels of NKA and AQP5 in nasal tissues from CRSwNP patients compared to control subjects, especially in eosinophilic CRSwNP. Furthermore, NKA mRNA levels in HNECs were downregulated by staphylococcal enterotoxin B (SEB), lipopolysaccharides (LPSs), inflammatory cytokine (IFN)-γ, IL-4, IL-13, and IL-1β. <b><i>Conclusion:</i></b> NKA and AQP5 expressions were decreased in CRSwNP. NKA in HNECs could be suppressed by SEB, LPS, IFN-γ, IL-4, IL-13, and IL-1β. Impairment of NKA may contribute to the genesis and development of CRSwNP via inducing AQP5 downregulation and edema.

scholarly journals MiR-133b Improves Decidualization of Endometrial Stromal Cells by Targeting KLF12 in Recurrent Implantation Failure

2021 ◽  
Author(s):  
Fenglin Mei ◽  
Chengcai Kong ◽  
Yan Wang ◽  
Jing Zhuang ◽  
Pingping Xue ◽  
...  
Keyword(s):  
Western Blot ◽  
Stromal Cells ◽  
Luciferase Reporter ◽  
Implantation Failure ◽  
Reporter System ◽  
Recurrent Implantation Failure ◽  
Endometrial Stromal Cells ◽  
Protein Levels ◽  
Qrt Pcr

Abstract Purpose Impaired decidualization contributes to the infertility in recurrent implantation failure (RIF). Herein, we focused on the function and probable mechanisms of miR-133b in endometrial stromal cells decidualization.Methods miR-133b and KLF12 protein levels in midsecretory endometrial tissues derived from women with and without RIF were measured by qRT-PCR and Western blot. Primary human endometrial stromal cells (HESCs) were isolated and cultured for in vitro decidualization assays. Luciferase reporter, qRT-PCR and Western blot assays were used to measure the relationship between miR-133b and KLF12.Results miR-133b was significantly downregulated, whereas KLF12 was upregulated in endometrial tissues from RIF. miR-133b effectively promoted HESCs in vitro decidualization through the modulation of KLF12 expression and the activation of LIF/STAT3 pathway. Conversely, inhibition of miR-133b expression reversed these effects. In addition, the luciferase reporter system demonstrated that miR-133b directly inhibited the expression of KLF12 by interacting with 3’ untranslated region of KLF12.Conclusion Our data suggest that miR-133b promotes HESCs decidualization by targeting KLF12 and reverses the impaired decidualization in RIF.

scholarly journals Demethylation of Secreted Frizzled-Related Protein2( SFRP2) Promoter Upregulates Wnt/β-Catenin Activity in Endometriosis

2021 ◽  
Author(s):  
Mei Yang ◽  
Chunfan Jiang ◽  
Lin Li ◽  
Xiaojie Huang ◽  
Hui Xing ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Luciferase Reporter ◽  
Gene Vector ◽  
Normal Endometrium ◽  
Protein Levels ◽  
Dnmt1 Expression ◽  
Ectopic Endometrium ◽  
Geo Database

Abstract Wnt/β-catenin signalling contributes to the metastasis and invasion in the etiology and pathogenesis of endometriosis (EMS), but why the WNT pathway is dysregulated in EMS remains unclear. This study aimed to explore the effects of demethylation of SFRP2 promoter on the Wnt/β-catenin activity in EMS. Aberrantly methylated-differentially expressed genes were identified from GEO database microarray data. 5 ectopic endometrium and 5 normal endometrium were get, subsequently, ectopic endometrium epithelial cells (EEECs) and normal endometrium epithelial cells (NEECs) were isolated in vitro. MSP, BSP, luciferase reporter assay, Lentivirus infection of high expression of SFRP2 gene vector, low expression of DNMT1 gene vector, and 5-Aza stimulation, RT-PCR and western blot were performed in the tissues or cells. It was found that compared with the normal endometrium and NEECs, the RNA and protein expression levels of SFRP2 were significantly increased while the SFRP2 promoter was demethylated in ectopic endometrium and EEECs. The 5-Aza treatment significantly upregulated SFRP2 mRNA and protein levels in EEECs. Furthermore, after the knockdown of DNMT1 expression, the demethylation of the SFRP2 promoter and upregulation of SFRP2 mRNA and protein in EEECs were observed. Meanwhile, the expression of lentivirus carrying SFRP2 cDNA up-regulates the activity of Wnt signaling and the protein expression of β-catenin in EEECs. In summary, the increased SFRP2 expression-induced Wnt/β-catenin signaling due to the demethylation of the SFRP2 promoter plays an important role in the pathogenesis of EMS, suggesting that SFRP2 might be a therapeutic target for EMS treatment.

scholarly journals miR-92 regulates the proliferation, migration, invasion and apoptosis of glioma cells by targeting neogenin

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 283-291
Author(s):  
Yi Wang ◽  
Yaohui Tian ◽  
Zonghao Li ◽  
Zhaoke Zheng ◽  
Liangliang Zhu
Keyword(s):  
Cell Proliferation ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter System ◽  
Pathological Mechanism ◽  
Glioma Cell Proliferation ◽  
Luciferase Reporter System ◽  
U87 Cells

AbstractThis study aimed to explore the pathological mechanism in regulating glioma progression. The expression of miR-92 and neogenin was evaluated by qRT-PCR and western blot. Cell viability and apoptosis were measured by MTT and flow cytometry assays, respectively. The migration and invasion abilities were examined by transwell assays. The interaction between miR-92 and neogenin was conducted by dual-luciferase reporter system. As a result, we found that the expression of miR-92 was up-regulated in glioma tissues and cell lines. Down-regulation of miR-92 inhibited glioma cell proliferation, migration, invasion and promoted cell apoptosis rate of U251 and U87 cells. Notably, miR-92 was identified to directly target to 3’-UTR of neogenin. Furthermore, neogenin was down-regulated in glioma tissues and cells in a miR-92-correlated manner. Overexpression of neigenin could cause similar results to miR-92 knockdown in U251 and U87 cells. However, the silencing of neogenin partially reversed the effects of miR-92 knockdown on cell proliferation, migration, invasion and apoptosis of glioma cells in vitro. In conclusion, we clarified that miR-92 knockdown could suppress the malignant progression of glioma cells in vitro by targeting neogenin. Therefore, miR-92 could serve as a potential diagnostic and prognostic marker in glioma patients

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