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1.
Adv Exp Med Biol ; 1131: 27-72, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646506

RESUMO

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.


Assuntos
Bioensaio , Canais Iônicos , Receptores Acoplados a Proteínas-G , Animais , Bioensaio/tendências , Descoberta de Drogas , Corantes Fluorescentes/metabolismo , Humanos , Canais Iônicos/análise , Receptores Acoplados a Proteínas-G/análise
2.
Zhongguo Zhong Yao Za Zhi ; 44(15): 3157-3161, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602867

RESUMO

In order to study the interaction between Pterocephalus hookeri and bitter taste receptors,three-dimensional structural models of bitter taste receptors TAS2 R16,TAS2 R14 and TAS2 R13 were established by homology modeling in this paper. Maestro software was used for docking the chemical constituents of P. hookeri with bitter taste receptors. The results showed that 25 chemical components of P. hookeri can regulate three bitter taste receptors. And these components were mainly iridoid glycosides and phenolic acids.This research focused on the comprehensive application of homology modeling and molecular docking technology to explore the interaction between bitter chemical constituents of P. hookeri and bitter taste receptors. This study provided assistance in revealing pharmacodynamic basis of bitter Tibetan medicine at molecular level. It also provided new ideas and methods for the study of Tibetan medicine.


Assuntos
Caprifoliaceae/química , Medicina Tradicional Tibetana , Simulação de Acoplamento Molecular , Receptores Acoplados a Proteínas-G/metabolismo , Correlação de Dados , Humanos , Paladar
3.
Curr Top Med Chem ; 19(16): 1436-1444, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31512997

RESUMO

Type 2 diabetes is a major health issue worldwide with complex metabolic and endocrine abnormalities. Hyperglycemia, defects in insulin secretion and insulin resistance are classic features of type 2 diabetes. Insulin signaling regulates metabolic homeostasis by regulating glucose and lipid turnover in the liver, skeletal muscle and adipose tissue. Major treatment modalities for diabetes include the drugs from the class of sulfonyl urea, Insulin, GLP-1 agonists, SGLT2 inhibitors, DPP-IV inhibitors and Thiazolidinediones. Emerging antidiabetic therapeutics also include classes of drugs targeting GPCRs in the liver, adipose tissue and skeletal muscle. Interestingly, recent research highlights several shared intermediates between insulin and GPCR signaling cascades opening potential novel avenues for diabetic drug discovery.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Descoberta de Drogas , Hipoglicemiantes/farmacologia , Receptor de Insulina/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hipoglicemiantes/química , Receptor de Insulina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
5.
BMC Evol Biol ; 19(1): 176, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470793

RESUMO

BACKGROUND: Vomeronasal type 1 receptor genes (V1Rs) are expected to detect intraspecific pheromones. It is believed that rodents rely heavily on pheromonal communication mediated by V1Rs, but pheromonal signals are thought to be confined in subterranean rodents that live in underground burrows. Thus, subterranean rodents may show a contrasting mode of V1R evolution compared with their superterranean relatives. RESULTS: We examined the V1R evolution in subterranean rodents by analyzing currently available genomes of 24 rodents, including 19 superterranean and 5 subterranean species from three independent lineages. We identified a lower number of putatively functional V1R genes in each subterranean rodent (a range of 22-40) compared with superterranean species (a range of 63-221). After correcting phylogenetic inertia, the positive correlation remains significant between the small V1R repertoire size and the subterranean lifestyle. To test whether V1Rs have been relaxed from functional constraints in subterranean rodents, we sequenced 22 intact V1Rs in 29 individuals of one subterranean rodent (Spalax galili) from two soil populations, which have been proposed to undergo incipient speciation. We found 12 of the 22 V1Rs to show significant genetic differentiations between the two natural populations, indicative of diversifying selection. CONCLUSION: Our study demonstrates convergent reduction of V1Rs in subterranean rodents from three independent lineages. Meanwhile, it is noteworthy that most V1Rs in the two Spalax populations are under diversifying selection rather than relaxed selection, suggesting that functional constraints on these genes may have retained in some subterranean species.


Assuntos
Evolução Molecular , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Odorantes/metabolismo , Spalax/genética , Animais , Feromônios/metabolismo , Filogenia , Receptores Acoplados a Proteínas-G/genética , Receptores Odorantes/genética , Seleção Genética , Spalax/classificação , Spalax/fisiologia , Órgão Vomeronasal/metabolismo
6.
Cell Host Microbe ; 26(2): 160-162, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31415748

RESUMO

Host-microbiome interactions affect host physiology, but the underlying mechanisms are not well understood. Recent papers from Chen et al. (2019) and Colosimo et al. (2019) in this issue of Cell Host & Microbe demonstrate that metabolites produced by several members of the gut microbiota can efficiently activate host G protein-coupled receptors and influence host physiology.


Assuntos
Microbiota , Receptores Acoplados a Proteínas-G , Bactérias , Humanos , Ligantes , Transdução de Sinais
7.
Biol Res ; 52(1): 44, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426858

RESUMO

BACKGROUND: Free fatty acid receptor 1 (FFAR1) is G-protein coupled receptor predominantly expressed in pancreatic ß-cells that is activated by a variety of free fatty acids (FFAs). Once activated, it promotes glucose-stimulated insulin secretion (GSIS). However, increased levels of FFAs lead to lipotoxicity, inducing loss of ß-cell function. FFAR1 plays a key role in the development of type 2 diabetes (T2D), and previous studies have indicated the importance of developing anti-diabetic therapies against FFAR1, although its role in the regulation of ß-cell function remains unclear. The present study investigated the role of FFAR1 under lipotoxic conditions using palmitic acid (PA). The rat insulinoma 1 clone 832/13 (INS-1 832/13) cell line was used as a model as it physiologically resembles native pancreatic ß-cells. Key players of the insulin signaling pathway, such as mTOR, Akt, IRS-1, and the insulin receptor (INSR1ß), were selected as candidates to be analyzed under lipotoxic conditions. RESULTS: We revealed that PA-induced lipotoxicity affected GSIS in INS-1 cells and negatively modulated the activity of both IRS-1 and Akt. Reduced phosphorylation of both IRS-1 S636/639 and Akt S473 was observed, in addition to decreased expression of both INSR1ß and FFAR1. Moreover, transient knockdown of FFAR1 led to a reduction in IRS-1 mRNA expression and an increase in INSR1ß mRNA. Finally, PA affected localization of FFAR1 from the cytoplasm to the perinucleus. CONCLUSIONS: In conclusion, our study suggests a novel regulatory involvement of FFAR1 in crosstalk with mTOR-Akt and IRS-1 signaling in ß-cells under lipotoxic conditions.


Assuntos
Células Secretoras de Insulina/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Linhagem Celular , Células Secretoras de Insulina/metabolismo , Ratos , Transdução de Sinais
8.
Clin Exp Rheumatol ; 37 Suppl 119(4): 69-75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31365333

RESUMO

OBJECTIVES: Relaxin is a potent anti-fibrotic hormone that has been tested to ameliorate fibrosis in systemic sclerosis (SSc), but with controversial results. The aim of the study is to sequence relaxin receptor gene RXFP1 and to assess its mRNA expression and protein levels in the skin of SSc patients and healthy subjects. METHODS: Fibroblasts were isolated from unaffected/affected skin samples of (n=16) limited-cutaneous-SSc-(LcSSc) and from affected ones of (n=4) diffuse-cutaneous-SSc-(DcSSc) patients. Fibroblasts from healthy subjects were used as controls. Sequencing of exonic target regions of interest for RXFP1 gene was performed, coupled with mRNA transcript variant analysis. RXFP1 mRNA and protein levels were assessed by quantitative-real-time-PCR-(qRT-PCR) and by immunocytochemistry-(ICC). Alpha-smooth-muscle-actin-(α-SMA) synthesis induced by transforming-growth-factor-beta-1-(TGF-ß1) stimulation was investigated in all fibroblasts with and without pre-treatment with serelaxin (a recombinant form of human relaxin-2 targeting the receptor RXFP1). RESULTS: Sequencing of RXFP1 gene showed no relevant mutations in all fibroblast populations. The analysis of mRNA transcripts revealed the presence of 13 different mRNA isoforms of RXFP1 (7 coding and 6 non-coding) upregulated in LcSSc/DcSSc-affected samples and not in LcSSc-unaffected and in healthy ones. On the contrary, ICC demonstrated the absence of RXFP1 in LcSSc/DcSSc-affected fibroblasts and the presence in LcSSc-unaffected and in healthy ones. To prove these findings, serelaxin pre-incubation was unable to counteract TGF-ß1-driven upregulation of α-SMA in LcSSc/DcSSc-affected fibroblasts only, but not in LcSSc-unaffected and healthy ones. CONCLUSIONS: The absence/altered expression of relaxin receptor RXFP1 in the affected fibroblasts of SSc patients could explain the inefficacy of relaxin-based anti-fibrotic treatments in the disease.


Assuntos
Fibroblastos/metabolismo , Relaxina , Esclerodermia Difusa , Escleroderma Sistêmico , Idoso , Feminino , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes , Relaxina/metabolismo , Esclerodermia Difusa/metabolismo , Esclerodermia Difusa/patologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia
9.
Medicine (Baltimore) ; 98(35): e16576, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464892

RESUMO

OBJECTIVES: G protein-coupled receptor 137 (GPR137) was reported to be associated with several cancers, but its role in bladder cancer has not been reported. The purpose of this study was to evaluate clinical significance of GPR137 in bladder cancer. METHODS: The expressions of GPR137 in pathological tissues and corresponding normal tissues from bladder cancer patients were detected via quantitative real time polymerase chain reaction (qRT-PCR). Western blot was performed to detect GPR137 expression in bladder cancer tissues and adjacent normal tissues. Chi-Squared test analyzed the relationship between GPR137 expression and clinical features of bladder cancer patients. Additionally, Kaplan-Meier method was adopted in estimating overall survival of bladder cancer patients. Prognostic value of GPR137 was evaluated through Cox regression analysis. RESULTS: The expression of GPR137 mRNA and protein in pathological tissues was significantly higher than that in adjacent normal tissues (P < .001). Moreover, similar result was found for bladder cancer patients and healthy controls (P < .001). And GPR137 expression was associated with tumor size (P = .006) and TNM stage (P = .012). The results of Kaplan-Meier analysis suggested that patients with high expression of GPR137 had shorter overall survival time than those with low expression (Log rank test, P = .001). Cox regression analysis indicated that GPR137 could act as an independent biomarker for bladder cancer prognosis (HR = 1.850, 95% CI = 1.272-2.689, P = .001). CONCLUSION: Abnormal expression of GPR137 is associated with bladder cancer and GPR137 is a potential biomarker for the therapy and prognosis of bladder cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Regulação para Cima , Neoplasias da Bexiga Urinária/metabolismo , Biomarcadores Tumorais/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Acoplados a Proteínas-G/genética , Análise de Sobrevida , Carga Tumoral , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia
10.
Sheng Li Xue Bao ; 71(4): 527-536, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440749

RESUMO

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.


Assuntos
Isquemia Encefálica , Estresse do Retículo Endoplasmático , Neurônios/citologia , Receptores Estrogênicos/fisiologia , Receptores Acoplados a Proteínas-G/agonistas , Traumatismo por Reperfusão , Animais , Apoptose , Região CA1 Hipocampal/citologia , Caspase 12/metabolismo , Caspase 3/metabolismo , Feminino , Proteínas de Choque Térmico/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fator de Transcrição CHOP/metabolismo
11.
Nat Commun ; 10(1): 2961, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273197

RESUMO

Persistent inflammation is a hallmark of many human diseases, including anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and atherosclerosis. Here, we describe a dominant trigger of inflammation: human serum factor H-related protein FHR1. In vitro, this protein selectively binds to necrotic cells via its N-terminus; in addition, it binds near necrotic glomerular sites of AAV patients and necrotic areas in atherosclerotic plaques. FHR1, but not factor H, FHR2 or FHR3 strongly induces inflammasome NLRP3 in blood-derived human monocytes, which subsequently secrete IL-1ß, TNFα, IL-18 and IL-6. FHR1 triggers the phospholipase C-pathway via the G-protein coupled receptor EMR2 independent of complement. Moreover, FHR1 concentrations of AAV patients negatively correlate with glomerular filtration rates and associate with the levels of inflammation and progressive disease. These data highlight an unexpected role for FHR1 during sterile inflammation, may explain why FHR1-deficiency protects against certain diseases, and identifies potential targets for treatment of auto-inflammatory diseases.


Assuntos
Proteínas Inativadoras do Complemento C3b/metabolismo , Inflamassomos/metabolismo , Monócitos/metabolismo , Monócitos/patologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Proteína C-Reativa/metabolismo , Proteínas do Sistema Complemento/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas Imobilizadas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Lipoproteínas LDL/metabolismo , Malondialdeído/metabolismo , Modelos Biológicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necrose , Ligação Proteica , Receptores Acoplados a Proteínas-G/metabolismo , Soro/metabolismo , Fosfolipases Tipo C/metabolismo
12.
BMC Evol Biol ; 19(1): 143, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299890

RESUMO

BACKGROUND: Our laboratory identified ADGRL4/ELTD1, an orphan GPCR belonging to the adhesion GPCR (aGPCR) family, as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. Little is known about how ADGRL4/ELTD1 (and aGPCRs in general) function, a problem compounded by a lack of known ligands or means of activation. With this in mind, we turned to computational evolutionary biology with the aim of better understanding ADGRL4/ELTD1. RESULTS: We identified ADGRL4/ELTD1 as a highly conserved early angiogenic gene which emerged in the first true vertebrates (bony fish) approximately 435 million years ago (mya), evolving alongside key angiogenic genes VEGFR2 and DLL4. We identified 3 evolutionary ADGRL4/ELTD1 variants based on EGF domain deletions with variant 2 first emerging 101 mya (95% CI 96-105) in Afrotheria and 82 mya (95% CI 76-89) in Primates. Additionally, conservation mapping across all orthologues reveals highest level conservation in EGF Ca binding domain 1, suggesting that this motif plays an essential role, as well as specific regions of the GAIN domain, GPS motif and 7TM domain, suggesting possible activation mechanisms and ligand binding positions. Additionally, we found that ADGRL4/ELTD1 (a member aGPCR family 1) is possibly ancestral to members of aGPCR family 2. CONCLUSION: This work establishes ADGRL4/ELTD1's evolution, sheds light on its possible activation and ligand binding zones, and establishes the first temporal references for the emergence of ADGRL4/ELTD1 variants during vertebrate evolution. Our approach is applicable to the greater aGPCR family and opens up new avenues for future experimental work.


Assuntos
Sequência Conservada , Evolução Molecular , Neovascularização Fisiológica , Receptores Acoplados a Proteínas-G/genética , Vertebrados/genética , Animais , Adesão Celular , Sistemas de Liberação de Medicamentos , Peixes/genética , Humanos , Neovascularização Fisiológica/genética , Filogenia , Domínios Proteicos , Receptores Acoplados a Proteínas-G/química , Deleção de Sequência , Fatores de Tempo
13.
Nature ; 571(7765): 398-402, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292548

RESUMO

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche1,2. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α)3, and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.


Assuntos
Envelhecimento , Senescência Celular , Esterases/metabolismo , Mucosa Intestinal/patologia , Celulas de Paneth/metabolismo , Regeneração , Envelhecimento/fisiologia , Animais , Senescência Celular/fisiologia , Esterases/antagonistas & inibidores , Esterases/biossíntese , Feminino , Humanos , Mucosa Intestinal/fisiologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , PPAR alfa/metabolismo , Celulas de Paneth/patologia , Receptores Acoplados a Proteínas-G/metabolismo , Nicho de Células-Tronco , Células-Tronco/patologia , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt
14.
Psychiatr Danub ; 31(2): 249-255, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31291233

RESUMO

BACKGROUND: Posttraumatic stress disorder (PTSD) is an anxiety disorder caused by highly traumatic experiences. The aim of this study was to investigate the influence of single nucleotide polymorphisms (SNPs) in the neuropeptide S receptor 1 (NPSR1) and the glutamate decarboxylase 1(GAD1) gene on PTSD and its psychopathological aspects among individuals affected by the Balkan wars during the 90s. SUBJECTS AND METHODS: This study was conducted as part of the South Eastern Europe (SEE) study on molecular mechanisms of PTSD. It comprised 719 participants (539 males), including those with current PTSD, remitted PTSD and healthy volunteers. Psychometric evaluation was performed using the Mini International Neuropsychiatric Interview (M.I.N.I.), the Clinician Administrated PTSD Scale (CAPS) andthe Brief Symptom Inventory (BSI). We examined NPSR1 single nucleotide polymorphism (SNP) rs324981 and GAD1 variant rs3749034 genotypes. Case-control analyses were carried out using logistical regression to determine genotype differences between all patients that had either current or remitted PTSD and control individuals. To analyse the influence of the analysed SNPs on PTSD severity, we performed linear regression analyses with CAPS and BSI within each of the two patient groups separately. All of the calculations were performed for additive allelic, recessive, dominant and genotypic models. RESULTS: We observed a nominally significant association for the major allele (G) of GAD1 rs3749034 with an increased risk to develop PTSD in a case control analysis in the recessive model (P=0.0315, odds ratio=0.47, SE=0.35). In contrast, a nominally significant association of the minor allele (A) with higher CAPS scores was identified within the patient group with lifetime PTSD in the dominant model (P=0.0372, ß=6.29, SE=2.99). None of these results did withstand correction for multiple tests. No nominal significant results of GAD1 rs3749034 were found with regard to the intensity of psychological BSI symptoms. Case-control analyses of NPSR1 rs324981 revealed a nominally significant higher risk for homozygous T allele carriers to develop PTSD (P=0.0452) in the recessive model. On the other hand, the T allele showed a nominally significant association with higher BSI scores in patients suffering from lifetime PTSD in the recessive model (P=0.0434). Again, these results were not significant anymore after correction for multiple tests. No associations of NPSR1 rs324981 and CAPS score was identified. CONCLUSION: The findings of this study provide some evidence that the NPSR1 and GAD1 polymorphisms might play a role in the development of war-related PTSD and its related psychological expressions. Further research is needed to elucidate the interactions of specific gene variants and environmental factors in the development of PTSD.


Assuntos
Glutamato Descarboxilase/genética , Polimorfismo de Nucleotídeo Único/genética , Receptores Acoplados a Proteínas-G/genética , Transtornos de Estresse Pós-Traumáticos/genética , Alelos , Conflitos Armados/psicologia , Europa Oriental , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
15.
Chem Commun (Camb) ; 55(61): 8975-8978, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290492
17.
Cell Prolif ; 52(5): e12661, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31318114

RESUMO

OBJECTIVES: Circular RNAs (circRNAs) are non-coding RNAs, some of which are thought to be involved in gastric cancer development. Here, we examined the functions of circRNA hsa_circ_006100 in gastric cancer cells and an animal model of gastric cancer. MATERIALS AND METHODS: The expression of hsa_circ_006100, miR-195 and various functional genes was determined by quantitative RT-PCR. Cell viability, clone formation, apoptosis and cell migration/invasion abilities were analysed by the CCK-8 assay, crystal violet staining, Hoechst staining and Transwell assay, respectively. A tumour model was established by subcutaneously injecting tumour cells into nude mice. Levels of protein expression were analysed by Western blotting and immunohistochemistry. RESULTS: A bioinformatics analysis showed that miR-195 was negatively co-expressed with hsa_circ_006100. Patients with a high hsa_circ_006100 level or low miR-195 level had tumours with a high TNM stage, poor cellular differentiation and lymph node metastasis. miR-195 was targeted and inhibited by hsa_circ_006100. Overexpression of hsa_circ_006100 enhanced cellular viability and proliferation, while miR-195 suppressed hsa_circ_006100-enhanced cell growth and induced apoptosis in MGC-803 and AGS cells. Forced hsa_circ_006100 expression promoted the migration and invasion of MGC-803 and AGS cells, while those activities were inhibited by miR-195. Mechanistically, GPRC5A was predicted as a target of miR-195 and was upregulated in gastric cancer. A miR-195 inhibitor restored cell viability, proliferation, migration and invasion, and repressed apoptosis via GPRC5A. In vivo studies showed that knockdown of hsa_circ_006100 delayed tumour growth, reduced PCNA expression and upregulated miR-195 and BCL-2 expression which was restored by miR-195 inhibition due to GPRC5A/EGFR signalling, and changed the EMT phenotype in vivo. CONCLUSIONS: Hsa_circ_006100 functions as an oncogene in gastric cancer and exerts its effects via miR-195/GPRC5A signalling.


Assuntos
MicroRNAs/metabolismo , RNA/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Neoplasias Gástricas/patologia , Animais , Antagomirs/metabolismo , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA/antagonistas & inibidores , RNA/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais , Neoplasias Gástricas/metabolismo
18.
Nat Commun ; 10(1): 2993, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278269

RESUMO

Activated hepatic stellate cell (aHSC)-mediated liver fibrosis is essential to the development of liver metastasis. Here, we discover intra-hepatic scale-up of relaxin (RLN, an anti-fibrotic peptide) in response to fibrosis along with the upregulation of its primary receptor (RXFP1) on aHSCs. The elevated expression of RLN serves as a natural regulator to deactivate aHSCs and resolve liver fibrosis. Therefore, we hypothesize this endogenous liver fibrosis repair mechanism can be leveraged for liver metastasis treatment via enforced RLN expression. To validate the therapeutic potential, we utilize aminoethyl anisamide-conjugated lipid-calcium-phosphate nanoparticles to deliver plasmid DNA encoding RLN. The nanoparticles preferentially target metastatic tumor cells and aHSCs within the metastatic lesion and convert them as an in situ RLN depot. Expressed RLN reverses the stromal microenvironment, which makes it unfavorable for established liver metastasis to grow. In colorectal, pancreatic, and breast cancer liver metastasis models, we confirm the RLN gene therapy results in significant inhibition of metastatic progression and prolongs survival. In addition, enforced RLN expression reactivates intra-metastasis immune milieu. The combination of the RLN gene therapy with PD-L1 blockade immunotherapy further produces a synergistic anti-metastatic efficacy. Collectively, the targeted RLN gene therapy represents a highly efficient, safe, and versatile anti-metastatic modality, and is promising for clinical translation.


Assuntos
Terapia Genética/métodos , Cirrose Hepática Experimental/terapia , Neoplasias Hepáticas/terapia , Hepatopatia Gordurosa não Alcoólica/terapia , Relaxina/genética , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Linhagem Celular Tumoral/transplante , Progressão da Doença , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Camundongos , Nanopartículas/administração & dosagem , Nanopartículas/química , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Plasmídeos/genética , Receptores Acoplados a Proteínas-G/metabolismo , Relaxina/metabolismo , Resultado do Tratamento , Microambiente Tumoral/genética , Regulação para Cima
19.
Handb Exp Pharmacol ; 256: 265-282, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31267167

RESUMO

Chenodeoxycholic acid (CDCA), 3α,7α-dihydroxy-5ß-cholan-24-oic acid, is a primary bile acid generated in the liver from cholesterol. In liver cells CDCA is conjugated with glycine or taurine to form two bile salts, Glyco-CDCA and Tauro-CDCA, before being released into the bile ducts. In the intestine, CDCA is further metabolized to generate a 7ß epimer, i.e., the ursodeoxycholic acid (UDCA), or dehydroxylate to generate lithocolic acid (LCA). In humans, CDCA is the physiological ligand for the bile acid sensor farnesoid X receptor (FXR), while LCA is a potent agonist for a G protein-coupled receptor, known as GPBAR1 (TGR5). Along with UDCA, CDCA has been clinically used for the dissolution of gallbladder stones at doses ranging from 375 to 750 mg/day, with a success rate of 8 to 18%. Because the efficacy of CDCA was significantly lower than that of UDCA and 18-30% of patients developed significant side effects, the most frequent being diarrhea and a reversible increase in aminotransferases plasma levels, this application has lost its therapeutic relevance. Additionally, the combination of CDCA with UDCA, generally at doses of 5-10 mg/kg each, has failed to provide significant advantages over UDCA alone. In 2017, CDCA has been approved as an orphan indication for the treatment of patients with cerebrotendinous xanthomatosis (CTX), a rare autosomal recessive disorder caused by mutations of sterol 27-hydroxylase (CYP27A1) gene. Since CYP27A1 is essential for cholesterol breakdown, CTX patients develop abnormal lipid storage with increased plasma and tissue levels of cholestanol and very low/absent production of CDCA. CDCA is a potent inhibitor of CYP27A1, and early initiation of CDCA therapy, at doses up to 750 mg/day, is considered the standard medical therapy for CTX resulting in decreased plasma levels of cholestanol and stabilization of neurologic symptoms. Studies in CTX patients have also shown that CDCA might suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in the liver. Furthermore, CDCA promotes the release of glucagon-like peptide-1 (GLP-1) in diabetic patients, likely by activating GPBAR1.


Assuntos
Ácido Quenodesoxicólico/uso terapêutico , Xantomatose Cerebrotendinosa , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanol/sangue , Colesterol , Humanos , Fígado , Receptores Citoplasmáticos e Nucleares , Receptores Acoplados a Proteínas-G
20.
Handb Exp Pharmacol ; 256: 19-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31302759

RESUMO

The BA-responsive GPCRs S1PR2 and TGR5 are almost ubiquitously expressed in human and rodent tissues. In the liver, S1PR2 is expressed in all cell types, while TGR5 is predominately found in non-parenchymal cells. In contrast to S1PR2, which is mainly activated by conjugated bile acids (BAs), all BAs serve as ligands for TGR5 irrespective of their conjugation state and substitution pattern.Mice with targeted deletion of either S1PR2 or TGR5 are viable and develop no overt phenotype. In liver injury models, S1PR2 exerts pro-inflammatory and pro-fibrotic effects and thus aggravates liver damage, while TGR5 mediates anti-inflammatory, anti-cholestatic, and anti-fibrotic effects. Thus, inhibitors of S1PR2 signaling and agonists for TGR5 have been employed to attenuate liver injury in rodent models for cholestasis, nonalcoholic steatohepatitis, and fibrosis/cirrhosis.In biliary epithelial cells, both receptors activate a similar signaling cascade resulting in ERK1/2 phosphorylation and cell proliferation. Overexpression of both S1PR2 and TGR5 was found in human cholangiocarcinoma tissue as well as in CCA cell lines, where stimulation of both GPCRs resulted in transactivation of the epidermal growth factor receptor and triggered cell proliferation as well as increased cell migration and invasiveness.This chapter will focus on the function of S1PR2 and TGR5 in different liver cell types and summarizes current knowledge on the role of these receptors in liver disease models.


Assuntos
Ácidos e Sais Biliares/fisiologia , Receptores Acoplados a Proteínas-G/fisiologia , Animais , Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Fígado , Hepatopatias , Camundongos , Receptores de Lisoesfingolipídeo/fisiologia
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