ABSTRACT
BACKGROUND: Circular RNAs (circRNAs) play important roles in cancer progression and metastasis. However, the expression profiles and biological roles of circRNAs in non-small cell lung cancer (NSCLC) remain unclear. METHODS: In this study, we identified a novel circRNA, hsa_circ_0006834 (termed circ6834), in NSCLC by RNA-seq and investigated the biological role of circ6834 in NSCLC progression in vitro and in vivo. Finally, the molecular mechanism of circ6834 was revealed by tagged RNA affinity purification (TRAP), western blot, RNA immunoprecipitation, dual luciferase reporter gene assays and rescue experiments. RESULTS: Our results showed that circ6834 was downregulated in NSCLC tumor tissues and cell lines. Circ6834 overexpression inhibited NSCLC cell growth and metastasis both in vitro and in vivo, while circ6834 knockdown had the opposite effect. We found that TGF-ß treatment decreased circ6834 expression, which was associated with the QKI reduction in NSCLC cells and circ6834 antagonized TGF-ß-induced EMT and metastasis in NSCLC cells. Mechanistically, circ6834 bound to AHNAK protein, a key regulator of TGF-ß/Smad signaling, and inhibited its stability by enhancing TRIM25-mediated ubiquitination and degradation. In addition, circ6834 acted as a miRNA sponge for miR-873-5p and upregulated TXNIP gene expression, which together inactivated the TGF-ß/Smad signaling pathway in NSCLC cells. CONCLUSION: In conclusion, circ6834 is a tumor-suppressive circRNA that inhibits NSCLC progression by forming a negative regulatory feedback loop with the TGF-ß/Smad signaling pathway and represents a novel therapeutic target for NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Carrier Proteins , Cell Proliferation , Gene Expression Regulation, Neoplastic , Lung Neoplasms , MicroRNAs , RNA, Circular , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/metabolism , RNA, Circular/genetics , MicroRNAs/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Animals , Mice , Cell Line, Tumor , Carrier Proteins/genetics , Carrier Proteins/metabolism , Disease Progression , Cell Movement/genetics , Signal Transduction , Female , Transforming Growth Factor beta/metabolism , Male , Epithelial-Mesenchymal Transition/geneticsABSTRACT
tRNA-derived fragments (tRFs) are an emerging category of small non-coding RNAs that are generated from cleavage of mature tRNAs or tRNA precursors. The advance in high-throughput sequencing has contributed to the identification of increasing number of tRFs with critical functions in distinct physiological and pathophysiological processes. tRFs can regulate cell viability, differentiation, and homeostasis through multiple mechanisms and are thus considered as critical regulators of human diseases including cancer. In addition, increasing evidence suggest the extracellular tRFs may be utilized as promising diagnostic and prognostic biomarkers for cancer liquid biopsy. In this review, we focus on the biogenesis, classification and modification of tRFs, and summarize the multifaceted functions of tRFs with an emphasis on the current research status and perspectives of tRFs in cancer.
Subject(s)
Neoplasms , RNA, Transfer , Humans , RNA, Transfer/genetics , Neoplasms/genetics , Neoplasms/pathologyABSTRACT
Lung cancer is the leading cause of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have emerged as key regulators of cancer development and progression, and as promising biomarkers for the diagnosis and prognosis of cancer. In this study, we identified a new lncRNA (LINC02159) that was upregulated in the tumor tissues and serum of non-small cell lung cancer (NSCLC) patients. We demonstrated that knockdown of LINC02159 inhibited NSCLC cell proliferation, migration, and invasion, but induced cell apoptosis and cell cycle arrest in vitro and retarded tumor growth in vivo, while overexpression of LINC02159 led to the opposite effect. We discovered that LINC02159 was highly correlated with cancer growth and metastasis-related pathways by using transcriptomic analysis and that YAP1 was a potential target gene of LINC02159. Mechanistically, LINC02159 bound to the Aly/REF export factor (ALYREF) to enhance the stability of YAP1 messenger RNA (mRNA) via m5C modification, which led to the overexpression of YAP1 and the activation of the Hippo and ß-catenin signaling pathways in NSCLC cells. Rescue experiments showed that LINC01259 promoted NSCLC progression in a YAP1- and ALYREF-dependent manner. In conclusion, LINC02159 plays an oncogenic role in NSCLC progression by regulating ALYREF/YAP1 signaling, and it has the potential to be utilized as a diagnostic marker and therapeutic target for NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , RNA, Long Noncoding , Humans , Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , Cell Line, Tumor , Signal Transduction , RNA, Long Noncoding/metabolism , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Hepatocellular carcinoma (HCC) is one common cancer in the world. Previous studies have shown that miR-17 family members are elevated in most tumors and promote tumor progression. However, there is no comprehensive analysis of the expression and functional mechanism of the microRNA-17 (miR-17) family in HCC. The aim of this study is to comprehensively analyze the function of the miR-17 family in HCC and the molecular mechanism of its role. Bioinfoimatics analysis of the miR-17 family expression profile and its relationship to clinical significance using The Cancer Genome Atlas (TCGA) database, and this result was confirmed using quantitative real-time polymerase chain reaction. miR-17 family members were tested for functional effects through transfection of miRNA precursors and inhibitors, and monitoring cell viability and migration by cell count and wound healing assays. In addition, we using dual-luciferase assay and Western blot demonstrated the targeting relationship between the miRNA-17 family and RUNX3. These members of miR-17 family were highly expressed in HCC tissues, and the overexpression of the miR-17 family promoted the proliferation and migration of SMMC-7721 cells, whereas treatment with anti-miR17 inhibitors caused the opposite effects. Notably, we also found that inhibitors anti-each member of miR-17 can suppress the expression of the entire family member. In addition, they can bind to the 3' untranslated region of RUNX3 to regulate its expression at the translational level. Our results proved that miR-17 family has oncogenic characteristics, overexpression every member of the family contributed to HCC cell proliferation and migration by reducing the translation of RUNX3.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , MicroRNAs , Humans , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation/genetics , MicroRNAs/genetics , Cell Movement/genetics , Gene Expression Regulation, Neoplastic , Core Binding Factor Alpha 3 Subunit/genetics , Core Binding Factor Alpha 3 Subunit/metabolismABSTRACT
The posttranscriptional control of gene expression mediated by RNA-binding proteins (RBPs) is essential to determine tumor cell fate. HuR is an RBP with increased expression in various cancer types. This study aimed to clarify the regulatory mechanism of HuR's contribution to breast cancer (BC) cell proliferation by inducing RAB5C expression. First, we analyzed the expression level of HuR and RAB5C in BC tissues and cell lines by immunohistochemistry, qRT-PCR, and western blot. Next, to further investigate the effect of HuR on RAB5C expression, we used short hairpin RNAs (shRNAs) to silence endogenous HuR expression in BC cell lines MCF7 and MDA-MB-231. The binding site of RAB5C mRNA and HuR was confirmed by RNA immunoprecipitation. Finally, the function of RAB5C was investigated using flow cytometry, colony formation, and MTT assays. We found that the expression of HuR and RAB5C was significantly upregulated in BC tissues and MCF-7 and MDA-MB231 cell lines. Importantly, RAB5C mRNA stability was increased through binding of HuR to its 3'UTR. Inhibition of HuR expression using shRNA decreased RAB5C mRNA, suggesting that HuR plays a role in regulating RAB5C expression level. In addition, suppression of RAB5C expression reduced BC cell growth. These results suggest RAB5C functions as an oncogene in BC cells, HuR promoted BC cell survival by facilitating RAB5C expression. Our findings suggest that HuR and RAB5C play important roles in BC cell survival.
Subject(s)
Breast Neoplasms , ELAV-Like Protein 1 , Female , Humans , 3' Untranslated Regions , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , ELAV-Like Protein 1/genetics , ELAV-Like Protein 1/metabolism , Gene Expression Regulation, Neoplastic , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolismABSTRACT
Aquaporins are transmembrane channels that allow for the passive permeation of water and other small molecules across biological membranes. Their channel activities are sensitive to mercury ions. Intriguingly, while most aquaporins are inhibited by mercury ions, several aquaporins are activated by mercury ions. The molecular basis of the opposing aquaporin regulation by mercury remains poorly understood. Herein, we investigated AqpZ inhibition and AQP6 activation upon binding of mercury ions using solid-state NMR (ssNMR) and molecular dynamics (MD) simulations. Based on the structure of the Hg-AqpZ complex constructed by MD simulations and ssNMR, we identified that the pore closure was caused by mercury-induced conformational changes of the key residue R189 in the selectivity filter region, while pore opening was caused by conformational changes of residues H181 and R196 in the selectivity filter region in AQP6. Both conformational changes were caused by the disruption of the H-bond network of R189/R196 by mercury. The molecular details provided a structural basis for mercury-mediated functional changes in aquaporins.
Subject(s)
MercuryABSTRACT
BACKGROUND: Increasing studies suggest that circular RNAs (circRNAs) are critical regulators of cancer development and progression. However, the biological roles and mechanisms of circRNAs in gastric cancer (GC) remain largely unknown. METHODS: We identified the differentially expressed circRNAs in GC by analyzing Gene Expression Omnibus (GEO) datasets. We explored the biological roles of circRNAs in GC by in vitro functional assays and in vivo animal studies. We performed tagged RNA affinity purification (TRAP), RNA immunoprecipitation (RIP), mass spectrometry (MS), RNA sequencing, luciferase reporter assays, and rescue experiments to investigate the mechanism of circRNAs in GC. RESULTS: Downregulated expression of circular RNA EIF4G3 (circEIF4G3; hsa_circ_0007991) was found in GC and was associated with poor clinical outcomes. Overexpression of circEIF4G3 suppressed GC growth and metastasis through the inhibition of ß-catenin signaling, whereas knockdown of circEIF4G3 showed the opposite effects. Mechanistic studies revealed that circEIF4G3 bound to δ-catenin protein to promote its TRIM25-mediated ubiquitin degradation and interacted with miR-4449 to upregulate SIK1 expression. CONCLUSION: Our findings uncovered a tumor suppressor function of circEIF4G3 in GC through the regulation of δ-catenin protein stability and miR-4449/SIK1 axis. CircEIF4G3 may act as a promising prognostic biomarker and therapeutic target for GC.
Subject(s)
MicroRNAs , Stomach Neoplasms , Animals , Catenins , Cell Line, Tumor , Cell Proliferation/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , Stomach Neoplasms/pathology , Ubiquitin , beta Catenin/genetics , Delta CateninABSTRACT
Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging technique to investigate the structures and dynamics of membrane proteins in an artificial or native membrane environment. However, the structural studies of proteins by ssNMR are usually prolonged or impeded by signal assignments, especially the assignments of signals for collection of distance restraints, because of serious overlapping of signals in 2D 13C-13C spectra. Sparse labeling of 13C spins is an effective approach to simplify the 13C spectra and facilitate the extractions of distance restraints. Here, we propose a new reverse labeling combination of six types of amino acid residues (Ile, Leu, Phe, Trp, Tyr and Lys), and show a clean reverse labeling effect on a model membrane protein E. coli aquaporin Z (AqpZ). We further combine this reverse labeling combination and alternate 13C-12C labeling, and demonstrate an enhanced dilution effect in 13C-13C spectra. In addition, the influences of reverse labeling on the labeling of the other types of residues are quantitatively analyzed in the two strategies (1, reverse labeling and 2, reverse labeling combining alternate 13C-12C labeling). The signal intensities of some other types of residues in 2D 13C-13C spectra are observed to be 20-50% weaker because of the unwanted reverse labeling. The extensively sparse 13C labeling proposed in this study is expected to be useful in the collection of distance restraints using 2D 13C-13C spectra of membrane proteins.
Subject(s)
Aquaporins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Carbon IsotopesABSTRACT
BACKGROUND: Increasing evidence indicates that circular RNAs (circRNAs) are dysregulated in human cancers. The biological roles of circRNAs in gastric cancer (GC) have not been well-characterized. METHODS: The GEO database was used to analyze circRNA expression profile in GC. The expression level of target circRNA in tumor tissues and adjacent non-tumor tissues was detected by reverse transcription-quantitative PCR. Gene transfection was used to manipulate the expression of circRNAs. The biological roles of circRNAs in cell proliferation, migration, and invasion were determined by cell counting, colony formation, transwell migration, Matrigel invasion, and mouse xenograft tumor assays. The interactions between circRNAs and miRNAs were verified by RNA immunoprecipitation and luciferase reporter assays. RESULTS: We found that circHN1 was upregulated in GC tissues and cell lines compared to adjacent non-tumor tissues and normal gastric epithelial cells. Additionally, circHN1 silencing significantly promoted GC cell growth, colony formation, migration, and invasion, whereas circHN1 overexpression had the opposite effects. CircHN1 overexpression also suppressed gastric cancer growth in the mouse xenograft tumor model. CircHN1 was mainly localized in the cytoplasm of GC cells and could bind to AGO2. MiR-1248 and miR-375 were predicted to interact with circHN1 by bioinformatic analyses. MiR-1248 and miR-375 overexpression inhibited the activity of the circHN1 luciferase reporter. CONCLUSION: CircHN1 is aberrantly expressed in GC and affects the proliferation and migration of gastric cancer cells by acting as miRNA sponge.
Subject(s)
Cell Movement/genetics , RNA, Circular/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Animals , Base Sequence , Cell Line, Tumor , Cell Proliferation/genetics , Exons/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Mice, Inbred BALB C , MicroRNAs/genetics , MicroRNAs/metabolism , Middle Aged , Neoplasm Invasiveness , RNA Splicing/genetics , RNA, Circular/genetics , Up-Regulation/geneticsABSTRACT
By integrating imaging and drug-delivery in a single system, fluorescent nano-multifunctional imaging platforms can offer simultaneous diagnosis and therapy to diseases like cancer. However, the synthesis of such system involves a tedious, time-consuming, and multi-step process. Herein we report a facile method based on simple ultrasonication to synthesize highly cross-linked, monodispersed fluorescent polyphosphazene nanoparticles from hexachlorocyclotriphosphazene (HCCP) and dichlorofluorescein (FD). Various functional groups (folic acid, PEG-NH2, and methylene blue) can be "fastened" in situ onto the poly(cyclotriphosphazene-co-dichlorofluorescein) (PCTPDF) nanoparticles to expand its application as nano-multifunctional platform. All the nanoparticles were characterized spectrophotometrically, and morphology was established by the images obtained from scanning electron microscope (SEM). The synthesized multifunctional nanoparticles exhibited low toxicity and penetrated through the cytomembranes of human colon cancer (HCT 116) cells. When applied to in vivo tumor imaging using biologically engineered mouse model, methylene blue functionalized (PCTPDF@MB) nanoparticles exhibited excellent photodynamic activity and imaging ability. Thus, PCTPDF nanoplatform based on multi-functional fluorescent nanoparticles might offer an efficient solution to new age theranostics. Apart from diagnostics application, PCTPDF, as a nanoplatform, could also be utilized to achieve more comprehensive application in modern analytic chemistry. Graphical Abstract The table of contents.
Subject(s)
Nanoparticles/chemistry , Neoplasms/diagnostic imaging , Organophosphorus Compounds/chemical synthesis , Polymers/chemical synthesis , Animals , Fluorescence , Fluorescent Dyes/chemistry , HCT116 Cells , Humans , Mice , Mice, Nude , Microscopy, Confocal , Microscopy, Electron, Scanning , Neoplasms/drug therapy , Organophosphorus Compounds/chemistry , Photochemotherapy , Polymers/chemistry , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Theranostic NanomedicineABSTRACT
Due to its simplicity, versatility, and high efficiency, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has emerged as one of the most promising approaches for treatment of a variety of genetic diseases, including human cancers. However, further translation of CRISPR/Cas9 for cancer gene therapy requires development of safe approaches for efficient, highly specific delivery of both Cas9 and single guide RNA to tumors. Here, novel core-shell nanostructure, liposome-templated hydrogel nanoparticles (LHNPs) that are optimized for efficient codelivery of Cas9 protein and nucleic acids is reported. It is demonstrated that, when coupled with the minicircle DNA technology, LHNPs deliver CRISPR/Cas9 with efficiency greater than commercial agent Lipofectamine 2000 in cell culture and can be engineered for targeted inhibition of genes in tumors, including tumors the brain. When CRISPR/Cas9 targeting a model therapeutic gene, polo-like kinase 1 (PLK1), is delivered, LHNPs effectively inhibit tumor growth and improve tumor-bearing mouse survival. The results suggest LHNPs as versatile CRISPR/Cas9-delivery tool that can be adapted for experimentally studying the biology of cancer as well as for clinically translating cancer gene therapy.
ABSTRACT
Transarterial chemoembolization (TACE) is the standard of care for treatment of intermediate hepatocellular carcinoma (HCC), however, key molecules involved in HCC cell survival and tumor metastasis post-TACE remain unclear. CD147 is a member of the immunoglobulin superfamily that is overexpressed on the surface of HCC cells and is associated with malignant potential and poor prognosis in HCC patients. In this study, using an Earle's Balanced Salt Solution medium culture model that mimics nutrient deprivation induced by TACE, we investigated the regulation of CD147 expression on HCC cells under starvation conditions and its functional effects on HCC cell death. During early stages of starvation, the expression of CD147 was considerably upregulated in SMMC7721, HepG2 and HCC9204 hepatoma cell lines at the protein levels. Downregulation of CD147 by specific small interfering RNA (siRNA) significantly promoted starvation-induced cell death. In addition, CD147 siRNA-transfected SMMC7721 cells demonstrated significantly increased levels of both apoptosis and autophagy as compared to cells transfected with control siRNA under starvation conditions, whereas no difference was observed between the two treatment groups under normal culture conditions. Furthermore, silencing of CD147 resulted in a remarkable downregulation of phosphorylated mammalian target of rapamycin (p-mTOR) in starved SMMC7721 cells. Finally, the combined treatment of starvation and anti-CD147 monoclonal antibody exhibited a synergistic HCC cell killing effect. Our study suggests that upregulation of CD147 under starvation may reduce hepatoma cell death by modulating both apoptosis and autophagy through mTOR signaling, and that CD147 may be a novel potential molecular target to improve the efficacy of TACE.
Subject(s)
Basigin/genetics , Gene Expression Regulation, Neoplastic , Gene Silencing , TOR Serine-Threonine Kinases/genetics , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Basigin/metabolism , Cell Line, Tumor , Culture Media/pharmacology , Hep G2 Cells , Humans , Phosphorylation/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
AIM: (5R)-5-Hydroxytriptolide (LLDT-8), an analogue of triptolide, displays lower toxicity compared to triptolide and has comparable immunosuppressive effects. We investigated the anti-inflammatory and neuroprotective effects of LLDT-8 on cerebral ischemia/reperfusion injury. METHODS: Nitric oxide production from microglia was assessed by measuring the nitrite concentration in the culture medium with Griess reagent. Microglial cells and ischemic brain tissues were examined for the expression of proinflammatory mediators by qPCR and western blot. Infarct volumes were assessed with TTC histology. The TLR4/NF-κB signaling pathway was analyzed with western blot and immunocytochemistry. RESULTS: LLDT-8 significantly reduced infarct sizes and expression of pro-inflammatory cytokines in the ischemic cortex. LLDT-8 inhibited NO release and expression of TNF-α, IL-1ß and iNOS in BV-2 microglia and primary microglia treated with LPS. In addition, LLDT-8 suppressed expression of TLR4, degradation of IκBα and nuclear translocation of NF-κB. CONCLUSION: LLDT-8 exerted anti-inflammatory effects and protected against acute cerebral ischemia/reperfusion injury possibly by acting through the IκB/NF-κB cascade to suppress microglia-mediated neuroinflammation.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Brain Ischemia/drug therapy , Diterpenes/therapeutic use , Neuroprotective Agents/therapeutic use , Reperfusion Injury/drug therapy , Stroke/drug therapy , Animals , Anti-Inflammatory Agents/pharmacology , Brain Ischemia/metabolism , Cell Line , Cell Survival/drug effects , Cells, Cultured , Diterpenes/pharmacology , Interleukin-1beta/genetics , Male , Mice, Inbred ICR , Microglia/drug effects , Microglia/metabolism , NF-KappaB Inhibitor alpha/metabolism , NF-kappa B/metabolism , Neuroprotective Agents/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Reperfusion Injury/metabolism , Signal Transduction/drug effects , Stroke/metabolism , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Over-activated microglia is involved in various kinds of neurodegenerative process including Parkinson, Alzheimer and HIV dementia. Suppression of microglial over activation has emerged as a novel strategy for treatment of neuroinflammation-based neurodegeneration. In the current study, anti-inflammatory and neuroprotective effects of the ent-kauranoid diterpenoids, which were isolated from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, were investigated in cultured microglia cells. Glaucocalyxin B (GLB), one of five ent-kauranoid diterpenoids, significantly decreased the generation of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in the lipopolysaccharide (LPS)-activated microglia cells. In addition, GLB inhibited activation of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS) in LPS-activated microglia cells. Furthermore, GLB strongly induced the expression of heme oxygenase (HO)-1 in BV-2 microglia cells. Finally, GLB exhibited neuroprotective effect by preventing over-activated microglia induced neurotoxicity in a microglia/neuron co-culture model. Taken together, the present study demonstrated that the GLB possesses anti-nueroinflammatory activity, and might serve as a potential therapeutic agent for treating neuroinflammatory diseases.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Diterpenes, Kaurane/pharmacology , Microglia/pathology , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/pathology , Neuroprotective Agents , Phytotherapy , Animals , Cells, Cultured , Cyclooxygenase 2/metabolism , Diterpenes, Kaurane/isolation & purification , Diterpenes, Kaurane/therapeutic use , Heme Oxygenase-1/metabolism , Interleukin-1beta/metabolism , Isodon/chemistry , Lipopolysaccharides/toxicity , Mice , Microglia/metabolism , Neurodegenerative Diseases/genetics , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Rats , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Matrix metalloproteinases (MMPs) are widely implicated in inflammation and tissue remodeling associated with various neurodegenerative diseases and play an important role in nociception and allodynia. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) plays a key regulatory role for MMP activities. However, the role of EMMPRIN in the development of neuropathic pain is not clear. Western blotting, real-time quantitative RT-PCR (qRT-PCR), and immunofluorescence were performed to determine the changes of messenger RNA and protein of EMMPRIN/OX47 and their cellular localization in the rat dorsal root ganglion (DRG) after nerve injury. Paw withdrawal threshold test was examined to evaluate the pain behavior in spinal nerve ligation (SNL) model. The lentivirus containing OX47 shRNA was injected into the DRG one day before SNL. The expression level of both mRNA and protein of OX47 was markedly upregulated in ipsilateral DRG after SNL. OX47 was mainly expressed in the extracellular matrix of DRG. Administration of shRNA targeted against OX47 in vivo remarkably attenuated mechanical allodynia induced by SNL. In conclusion, peripheral nerve injury induced upregulation of OX47 in the extracellular matrix of DRG. RNA interference against OX47 significantly suppressed the expression of OX47 mRNA and the development of mechanical allodynia. The altered expression of OX47 may contribute to the development of neuropathic pain after nerve injury.
Subject(s)
Basigin/metabolism , Ganglia, Spinal/metabolism , Hyperalgesia/metabolism , Spinal Nerves/injuries , Animals , Extracellular Matrix/metabolism , Male , Pain Threshold/physiology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Up-RegulationABSTRACT
CD4(+) FOXP3(+) regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4(+) CD25(high) CD127(low/-) Treg cells convert to two subpopulations with distinctive FOXP3(+) and FOXP3(-) phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events.
Subject(s)
Cell Differentiation , Cell Lineage , Cellular Reprogramming , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , Histones/metabolism , T-Lymphocytes, Regulatory/metabolism , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Proliferation , Cells, Cultured , Cellular Reprogramming/genetics , Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation , Forkhead Transcription Factors/genetics , Gene Expression Profiling/methods , Gene Expression Regulation , Genetic Markers , High-Throughput Nucleotide Sequencing , Humans , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-7 Receptor alpha Subunit/metabolism , Methylation , Phenotype , T-Lymphocytes, Regulatory/immunology , Th2 Cells/immunology , Th2 Cells/metabolism , Time FactorsABSTRACT
Glial activation-mediated neuroinflammation plays a pivotal role in the process of several neuroinflammatory diseases including stroke, Alzheimer's diseases, Parkinson's diseases, multiple sclerosis and ischemia. Inhibition of microglial activation may ameliorate neuronal degeneration under the inflammatory conditions. In the present study, a number of 5α-cholestan-6-one derivatives were prepared and the anti-inflammatory effects of these compounds were evaluated in LPS-stimulated BV-2 microglia cells. Those derivatives were synthesized from readily available hyodeoxycholic acid (1). Among the tested compounds, several analogs (16-18, 25, 35, 38) exhibited potent inhibitory activities on nitric oxide production with no or weak cell toxicity. Compound 16 also significantly suppressed the expression of TNF-α, interleukin (IL)-1ß, cyclooxygenase (COX-2) as well as inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 microglia cells. In addition, compound 16 markedly reduced infarction volume in a focal ischemic mice model.
Subject(s)
Cholestanones/pharmacology , Drug Discovery , Inflammation/drug therapy , Microglia/drug effects , Nitric Oxide/antagonists & inhibitors , Animals , Cell Line , Cell Survival/drug effects , Cholestanones/chemical synthesis , Cholestanones/chemistry , Crystallography, X-Ray , Disease Models, Animal , Dose-Response Relationship, Drug , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Mice , Microglia/metabolism , Models, Molecular , Molecular Structure , Nitric Oxide/biosynthesis , Structure-Activity RelationshipABSTRACT
Solid-state NMR is a powerful tool for studying membrane proteins in a native-like lipid environment. 3D magic angle spinning (MAS) NMR was employed to characterize the structure of E.coli diacylglycerol kinase (DAGK) reconstituted into its native E.coli lipid membranes. The secondary structure and topology of DAGK revealed by solid-state NMR are different from those determined by solution-state NMR and X-ray crystallography. This study provides a good example for demonstrating the influence of membrane environments on the structure of membrane proteins.
Subject(s)
Diacylglycerol Kinase/chemistry , Escherichia coli/enzymology , Crystallography, X-Ray , Diacylglycerol Kinase/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, SecondaryABSTRACT
Purpose: This study aims to investigate the impact of physical activity and self-control on college students' mobile phone addiction through cross-lagged longitudinal surveys, addressing the limitations of previous cross-sectional studies. Patients and methods: A total of 414 college students were tracked three times during a 12-month period using the Physical Activity Rating Scale-3 (PARS-3), the Mobile Phone Addiction Tendency Scale (MPATS), and the Brief Self-Control Scale (BSCS). AMOS25.0 software was used to construct the cross-lagged relationship model, and the maximum likelihood approach was employed to investigate the model fitting. The asynchronous correlation between variables was investigated from the time series through the cross-lagged path coefficient. Results: The fitting indexes of the cross-lagged model showed x 2/df = 5.098, GFI = 0.977, NFI = 0.969, IFI = 0.975, CFI = 0.974; RMSEA = 0.100, and SRMR = 0.030. The calculation conducted by combining the path coefficient of the model shows that PA and SC are the antecedent variables of MPA, and PA is the antecedent variable of SC. In addition, SC serves as a mediator in the path of PA, affecting MPA. Conclusion: (1) physical activity can positively affect subsequent self-control; (2) physical activity can negatively influence subsequent mobile phone addiction; (3) self-control can negatively affect subsequent mobile phone addiction; and (4) physical activity can indirectly influence subsequent mobile phone addiction through self-control.
ABSTRACT
Structure determination of membrane proteins in native cellular membranes is critical to precisely reveal their structures in physiological conditions. However, it remains challenging for solid-state nuclear magnetic resonance (ssNMR) due to the low sensitivity and high complexity of ssNMR spectra of cellular membranes. Here, we present the structure determination of aquaporin Z (AqpZ) by ssNMR in Escherichia coli inner membranes. To enhance the signal sensitivity of AqpZ, we optimized protein overexpression and removed outer membrane components. To suppress the interference of background proteins, we used a "dual-media" expression approach and antibiotic treatment. Using 1017 distance restraints obtained from two-dimensional 13C-13C spectra based on the complete chemical shift assignments, the 1.7-Å ssNMR structure of AqpZ is determined in E. coli inner membranes. This cellular ssNMR structure determination paves the way for analyzing the atomic structural details for membrane proteins in native cellular membranes.