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1.
Life Sci ; 244: 117306, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31953159

RESUMO

AIMS: Accumulated evidence indicates that the dysregulation of circular RNAs (circRNAs) plays pivotal roles in many human diseases including preeclampsia (PE). Circ_0063517 has been verified to be down-regulated in PE. But the role of circ_0063517 in PE is still unclear. This research aims to probe into the effect of circ_0063517 on angiogenesis in PE development. MAIN METHODS: The expression of circ_0063517, endothelin receptor type B (ETBR) and miR-31-5p was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR). MTT assay, colony formation assay, scratch assay, transwell assay, and tube formation assay were performed to detect proliferation, migration, and angiogenesis, respectively. Dual luciferase reporter system and RNA immunoprecipitation (RIP) assay were carried out to determine the interaction between miR-31-5p and circ_0063517(or ETBR). ETBR, VEGFRA, and VEGFR2 levels were detected by western blot analysis. The effect of circ_0063517 and ETBR on angiogenesis was evaluated in N-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced PE in vivo. KEY FINDINGS: The levels of circ_0063517 and ETBR were down-regulated in the placenta tissue of PE patients. Conversely, the level of miR-31-5p was up-regulated in PE. Overexpression of circ_0063517 or knockdown of miR-31-5p facilitated growth, migration, and angiogenesis of vascular endothelial cells. Circ_0063517 knockdown-induced repression of the expression of ETBR, VEGFA, and VEGFR2 was partly counteracted by ETBR overexpression. Mechanistically, circ_0063517 sponged miR-31-5p to regulate ETBR expression. Finally, circ_0063517 promoted angiogenesis via enhancing ETBR expression in PE in vivo. SIGNIFICANCE: Our findings suggest that circ_0063517-miR-31-5p-ETBR axis regulates angiogenesis during the pathological process of PE.


Assuntos
MicroRNAs/metabolismo , Pré-Eclâmpsia/metabolismo , Receptor de Endotelina B/metabolismo , Animais , Linhagem Celular , Proliferação de Células/fisiologia , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/genética , Neovascularização Patológica/genética , Pré-Eclâmpsia/genética , Gravidez , Ratos , Ratos Sprague-Dawley
2.
Gut ; 69(2): 329-342, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31439637

RESUMO

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Chaperonas de Histonas/fisiologia , Neoplasias Hepáticas/fisiopatologia , Estresse Oxidativo/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes/métodos , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Camundongos Endogâmicos BALB C , Camundongos Nus , Estresse Oxidativo/genética , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/biossíntese , Fatores de Elongação da Transcrição/genética , Regulação para Cima/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Cancer Sci ; 111(1): 200-208, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31778288

RESUMO

Integrins are transmembrane proteins that mediate cell adhesion to the extracellular matrix. Integrin α11 (ITGA11) is not expressed in normal alveolar epithelial cells and is a known receptor for collagen. While integrin α11ß1 overexpression in the tumor stroma has been associated with tumor growth and metastatic potential of non-small cell lung cancer (NSCLC), little is known about the role of ITGA11 in tumor cells. Thus, we examined the RNA expression of ITGA11 by quantitative RT-PCR in 80 samples collected from NSCLC patients who had undergone surgical resection and analyzed the clinical outcomes. We found that high expression of ITGA11 was associated with lower recurrence-free survival in all NSCLC patients (P = 0.043) and in stage I NSCLC patients (P = 0.049). These results were consistent with in silico analyses of the Cancer Genome Atlas database. We also analyzed cell proliferation, migration and invasion capacity in lung cancer cell lines after overexpression of ITGA11. Overexpression of ITGA11 in lung cancer cell lines had little effect on cell proliferation but resulted in increased migration and invasion capacity. Our findings suggest that ITGA11 plays a significant role in cancer migration and invasion, leading to higher recurrence. ITGA11 expression may be a predictor of poor prognosis in patients with surgically resected NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Cadeias alfa de Integrinas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Adulto , Idoso , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
4.
Gene ; 726: 144132, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669643

RESUMO

The NF-κB signaling pathway is a key regulator of CRC cell proliferation, apoptosis, angiogenesis, inflammation, metastasis, and drug resistance. Over-activation of the NF-κB pathway is a feature of colorectal cancer (CRC). While new combinatorial treatments have improved overall patient outcome; quality of life, cost of care, and patient survival rate have seen little improvement. Suppression of the NF-κB signaling pathway using biological or specific pharmacological inhibitors is a potential therapeutic approach in the treatment of colon cancer. This review summarizes the regulatory role of NF-κB signaling pathway in the pathogenesis of CRC for a better understanding and hence a better management of the disease.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Humanos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Qualidade de Vida , Taxa de Sobrevida
5.
Life Sci ; 242: 117229, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31887298

RESUMO

AIMS: Neutrophil elastase (NE) is a critical proteolytic enzyme that is involved in cancer. We previously reported high NE expression in peripheral blood neutrophils from acute promyelocytic leukemia (APL) patients. The present study aimed to elucidate the specific role and mechanisms of NE in APL development. MATERIALS AND METHODS: NE expression was detected in APL bone marrow samples and analyzed in the BloodSpot database. CCK-8 assay and flow cytometry were used to assess cell proliferation and cell cycle distribution, respectively. The expression levels of proliferation and differentiation markers were measured by Western blotting and quantitative real-time PCR. The co-expression and interaction of NE and p200 cut-like homeobox 1 (CUX1) were evaluated by indirect immunofluorescence, co-immunoprecipitation, and in situ proximity ligation assay. KEY FINDINGS: NE was highly expressed in APL bone marrow and blood neutrophils. NE overexpression promoted the proliferation and inhibited the differentiation of NB4 cells, whereas NE downregulation achieved the opposite results in U937 cells. Mechanistically, NE interacted with and effectively hydrolyzed the tumor suppressor p200 CUX1. Rescue experiments revealed that p200 CUX1 upregulation reversed the functional influence of NE on APL cells. SIGNIFICANCE: NE-mediated proteolysis of the tumor suppressor p200 CUX1 promotes APL progression. NE/p200 CUX1 axis is a novel and promising therapeutic target for APL treatment.


Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia Promielocítica Aguda/enzimologia , Elastase de Leucócito/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Adulto , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Células HL-60 , Proteínas de Homeodomínio/fisiologia , Humanos , Imunoprecipitação , Leucemia Promielocítica Aguda/metabolismo , Elastase de Leucócito/fisiologia , Masculino , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sincalida/metabolismo , Fatores de Transcrição/fisiologia , Células U937
6.
Gene ; 725: 144143, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31629816

RESUMO

Atherosclerosis is a common cardiovascular disorder and is characterized by damage of endothelial cells, cell inflammation, hyper-proliferation of vascular smooth muscle cells and the accumulation of extracellular lipids and fibrous tissues. In this study, we firstly examined the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) in homocysteine (Hcy)-stimulated human aortic smooth muscle cells (HASMCs), and then looked into the potential molecular signaling axis of linc-ROR in regulating the proliferation and migration of HASMCs. Hcy promoted HASMC proliferation and up-regulated linc-ROR expression. Functional studies showed that linc-ROR exerted enhanced actions on the proliferation and migration of HASMCs. In addition, linc-ROR acted as a competing endogenous RNA for miR-195-5p and repressed the miR-195-5p expression in HASMCs. Linc-ROR was up-regulated the miR-195-3p was down-regulated in the plasma from CAD patients when compared to normal controls. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a target of miR-195-5p and was negatively regulated by miR-195-5p in HASMCs. The rescue experiments revealed that linc-ROR-mediated HASMC proliferation and migration may be via regulating miR-195-5p/FGF2 axis. Linc-ROR inhibition blocked the miR-195-5p/FGF2 signaling in Hcy-treated HASMCs, and this effect may also involve in the miR-195-5p/FGF2 axis. To summarize, the data of the present study identified the up-regulation of linc-ROR in Hcy-stimulated HASMCs, and further mechanistic functional studies revealed that linc-ROR promoted HASMC proliferation and migration via regulating miR-195-5p/FGF2 axis. The present study provided the novel actions of linc-ROR in regulating HASMC proliferation and migration, which may be related to the pathophysiology of atherosclerosis.


Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Autofagia/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/fisiologia , Homocisteína/farmacologia , Humanos , MicroRNAs/genética , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Transdução de Sinais
7.
Biol Res ; 52(1): 59, 2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801629

RESUMO

OBJECTIVES: In varicose veins, vascular smooth muscle cells (VSMCs) often shows phenotypic transition and abnormal proliferation and migration. Evidence suggests the FOXC2-Notch pathway may be involved in the pathogenesis of varicose veins. Here, this study aimed to explore the role of long non-coding RNA FOXC2-AS1 (FOXC2 antisense RNA 1) in phenotypic transition, proliferation, and migration of varicose vein-derived VSMCs and to explore whether the FOXC2-Notch pathway was involved in this process. METHODS: The effect of FOXC2-AS1 on the proliferation and migration of human great saphenous vein smooth muscle cells (SV-SMCs) was analyzed using MTT assay and Transwell migration assay, respectively. The levels of contractile marker SM22α and synthetic marker osteopontin were measured by immunohistochemistry and Western blot to assess the phenotypic transition. RESULTS: The human varicose veins showed thickened intima, media and adventitia layers, increased synthetic VSMCs, as well as upregulated FOXC2-AS1 and FOXC2 expression. In vitro assays showed that FOXC2-AS1 overexpression promoted phenotypic transition, proliferation, and migration of SV-SMCs. However, the effect of FOXC2-AS1 overexpression could be abrogated by both FOXC2 silencing and the Notch signaling inhibitor FLI-06. Furthermore, FOXC2-AS1 overexpression activated the Notch pathway by upregulating FOXC2. CONCLUSION: FOXC2-AS1 overexpression promotes phenotypic transition, proliferation, and migration of SV-SMCs, at least partially, by activating the FOXC2-Notch pathway.


Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Miócitos de Músculo Liso/metabolismo , Veia Safena/metabolismo , Células Cultivadas , Humanos , Miócitos de Músculo Liso/patologia , Fenótipo , Veia Safena/patologia , Transdução de Sinais , Regulação para Cima
8.
Biol Res ; 52(1): 60, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31847887

RESUMO

BACKGROUND: Recent studies have confirmed that RASAL1 has an antitumor effect in many cancers, but its functional role and the molecular mechanism underlying in colon cancer has not been investigated. RESULTS: We collected human colon cancer tissues and adjacent non-tumor tissues, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and normal colonic mucosa cell line NCM460. RT-qPCR was used to detect the RASAL1 level in the clinical tissues and cell lines. In LoVo and HCT-116, RASAL1 was artificially overexpressed. Cell viability and proliferation were measured using CCK-8 assays, and cell cycle was detected via PI staining and flow cytometry analysis. RASAL1 significantly inhibited the cell proliferation via inducing cell cycle arrest, suppressed cell cycle associated protein expression, and decreased the lipid content and inhibited the SCD1 expression. Moreover, SCD1 overexpression induced and downregulation repressed cell proliferation by causing cell cycle arrest. Additionally, luciferase reporter assays were performed to confirm the direct binding between SREBP1c, LXRα and SCD1 promoter, we also demonstrated that RASAL1 inhibit SCD1 3'-UTR activity. RASAL1 inhibited tumor growth in xenograft nude mice models and shows inhibitory effect of SCD1 expression in vivo. CONCLUSION: Taken together, we concluded that RASAL1 inhibited colon cancer cell proliferation via modulating SCD1 activity through LXRα/SREBP1c pathway.


Assuntos
Proliferação de Células/fisiologia , Neoplasias do Colo/patologia , Proteínas Ativadoras de GTPase/metabolismo , Receptores X do Fígado/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Proteínas Ativadoras de GTPase/genética , Humanos , Receptores X do Fígado/genética , Camundongos , Estearoil-CoA Dessaturase/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética
9.
Immunity ; 51(6): 1119-1135.e5, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31757672

RESUMO

T cells play important multifaceted roles during dengue infection, and understanding their responses is important for defining correlates of protective immunity and identifying effective vaccine antigens. Using mass cytometry and a highly multiplexed peptide-HLA (human leukocyte antigen) tetramer staining strategy, we probed T cells from dengue patients-a total of 430 dengue and control candidate epitopes-together with key markers of activation, trafficking, and differentiation. During acute disease, dengue-specific CD8+ T cells expressed a distinct profile of activation and trafficking receptors that distinguished them from non-dengue-specific T cells. During convalescence, dengue-specific T cells differentiated into two major cell fates, CD57+ CD127--resembling terminally differentiated senescent memory cells and CD127+ CD57--resembling proliferation-capable memory cells. Validation in an independent cohort showed that these subsets remained at elevated frequencies up to one year after infection. These analyses aid our understanding of the generation of T cell memory in dengue infection or vaccination.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Antígenos HLA/imunologia , Adulto , Linfócitos B/imunologia , Antígenos CD57/metabolismo , Diferenciação Celular/imunologia , Proliferação de Células/fisiologia , Epitopos de Linfócito T/imunologia , Feminino , Antígenos HLA/classificação , Humanos , Memória Imunológica/imunologia , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade
10.
Invest Ophthalmol Vis Sci ; 60(14): 4820-4829, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31747682

RESUMO

Purpose: This study was aimed at investigating the association of long noncoding RNA (lncRNA)-related single nucleotide polymorphisms (SNPs) with Vogt-Koyanagi-Harada (VKH) disease. Methods: LncRNA-related SNPs were selected by multi-omics analysis. Genotyping, expression of lncRNA and mRNA, cell proliferation, and cytokine production were tested by MassARRAY System, real-time PCR, CCK8, and ELISA. Results: A significant association with VKH was found for lnc-TOR3A-1:1/rs3829794, which is located in a non-HLA region (CC genotype: Bonferroni corrected P values [PC] = 2.98 × 10-8, odds ratio [OR] = 0.62; TT genotype: PC = 1.64 × 10-8, OR = 1.57; C allele: PC = 1.39 × 10-12, OR = 0.71). Additionally, an association was found for four lncRNA SNPs located in the HLA region. Functional experiments in rs3829794 genotyped individuals showed decreased ABL2 (ABL proto-oncogene 2, nonreceptor tyrosine kinase) expression, decreased proliferation of anti-CD3 plus anti-CD28-stimulated peripheral blood mononuclear cells (PBMCs), and an increased production of IL-10 in CC carriers compared to TT carriers (P = 0.0073, P = 0.0011, and P = 0.002, respectively). Conclusions: Our study identified five new loci associated with VKH susceptibility and identified a functional variant (lnc-TOR3A-1:1/rs3829794) that confers risk for VKH, which is possibly mediated by modulating gene expression, proliferation of lymphocytes, and regulation of anti-inflammatory cytokine production.


Assuntos
Adenosina Trifosfatases/genética , Predisposição Genética para Doença/genética , Antígenos de Histocompatibilidade Menor/genética , Chaperonas Moleculares/genética , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética , Síndrome Uveomeningoencefálica/genética , Adulto , Estudos de Casos e Controles , Proliferação de Células/fisiologia , Células Cultivadas , Citocinas/metabolismo , Replicação do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Síndrome Uveomeningoencefálica/metabolismo , Síndrome Uveomeningoencefálica/patologia
11.
Invest Ophthalmol Vis Sci ; 60(14): 4670-4680, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31725166

RESUMO

Purpose: Long noncoding RNAs (lncRNAs) are important in disease progression and cellular functions. This study aimed to conduct global lncRNA profiling and characterize the role of lncRNA 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase delta 3-sence RNA 1 (PLCD3-OT1) in the progression of age-related cataract (ARC). Methods: We performed lncRNA expression profiling of lens capsule from ARC groups and age-matched groups using high-throughput RNA-sequencing. Real-time PCR was conducted to detect the expression pattern of lncRNA and mRNA in the clinical samples and cell model. Assays of cell-counting kit-8, 5'-ethynyl-2'-deoxyuridine, TUNEL, and propidium iodide staining were used to detect cell viability, proliferation, apoptosis, and cell cycle. We also performed fluorescence in situ hybridization assay to detect the location of lncRNA, and verified the endogenous competitive RNA mechanism between miRNAs, lncRNAs, and target genes via double-luciferase reporter analyses. Results: The expression of lncRNA PLCD3-OT1 and PLCD3 were significantly decreased in ARC. PLCD3-OT1 overexpression promoted the expression of PLCD3, cell viability, proliferation, and inhibited cell apoptosis upon oxidative stress, while knockdown of PLCD3 showed the opposite results. Mechanistically, PLCD3-OT1functions through positively regulation the expression of PLCD3. In addition, PLCD3-OT1 may act as a ceRNA to regulate the expression of PLCD3 through competition for miR-224-5p. Conclusions: PLCD3-OT1 and PLCD3 may become potential therapeutic targets for the prognosis, diagnosis, and treatment of ARC.


Assuntos
Catarata/prevenção & controle , MicroRNAs/metabolismo , Fosfolipase C delta/fisiologia , RNA Longo não Codificante/fisiologia , Idoso , Western Blotting , Catarata/metabolismo , Catarata/patologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Marcação In Situ das Extremidades Cortadas , Lentivirus/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Transfecção
12.
Invest Ophthalmol Vis Sci ; 60(14): 4748-4758, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31731295

RESUMO

Purpose: Lens fibrosis involves aberrant growth, migration, and transforming growth factorß (TGFß)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). In this study, we investigated the role of the bromo- and extra-terminal domain (BET) inhibitor in lens fibrotic disorder to identify drug-based therapies. Methods: Rat lens explants, rabbit primary lens epithelial cells (rLECs), human lens explants and human SRA01/04 cells were treated with TGFß2 in the presence or absence of the BET bromodomain inhibitor JQ1 or the MYC inhibitor 10058-F4. Proliferation was determined by MTS assay. Cell migration was measured by wound healing and transwell assays. The expression levels of fibronectin (FN), α-smooth muscle actin (α-SMA), E-cadherin, and phosphorylated downstream Smads were analyzed by Western blot, qRT-PCR, and immunocytochemical experiments. Transcriptome analysis was conducted to explore the molecular mechanism. Results: Blockage of BET bromodomains with JQ1 significantly suppressed rLECs proliferation by inducing G1 cell cycle arrest. Furthermore, JQ1 attenuated TGFß2-dependent upregulation of mesenchymal gene expression and phosphorylation of Smad2/3 during the progression of EMT, whereas E-cadherin expression was preserved. JQ1 repressed MYC expression, which was dose- and time-dependently upregulated by TGFß2. Inhibiting MYC with either the small-molecule inhibitor 10058-F4 or genetic knockdown phenocopied the effects of JQ1 treatment. MYC overexpression partially reversed the JQ1-regulated EMT-related alteration of gene expression. Both JQ1 and 10058-F4 blocked the expression of TGFß receptor II and integrin αv in rLECs and abolished TGFß2-induced opacification and subcapsular plaque formation in rat lens explants. Conclusions: Our results demonstrate the antifibrotic role of JQ1 in maintaining the epithelial characteristics of LECs and blocking TGFß2-induced EMT, possibly by downregulating MYC, thereby providing new avenues for treating lens fibrosis.


Assuntos
Azepinas/farmacologia , Regulação para Baixo , Células Epiteliais/efeitos dos fármacos , Cristalino/patologia , Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Triazóis/farmacologia , Actinas/metabolismo , Adulto , Animais , Western Blotting , Caderinas/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibronectinas/metabolismo , Fibrose/prevenção & controle , Humanos , Coelhos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Tiazóis/farmacologia , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Fator de Crescimento Transformador beta2/farmacologia
13.
Invest Ophthalmol Vis Sci ; 60(14): 4759-4773, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738824

RESUMO

Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.


Assuntos
Diferenciação Celular/fisiologia , Colinérgicos/farmacologia , Células Ependimogliais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/embriologia , Transdução de Sinais/fisiologia , Esferoides Celulares/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Embrião de Galinha , Colina O-Acetiltransferase/metabolismo , Crioultramicrotomia , Ácido Glutâmico/farmacologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neurônios Retinianos/citologia , Esferoides Celulares/metabolismo , Fixação de Tecidos , Vimentina/metabolismo
14.
Int J Sports Med ; 40(14): 921-930, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31614382

RESUMO

Myocardial damage due to dysfunctional myocardium has been increasing, and the prognosis of pharmacological and device-based therapies remain poor. Isl1-expressing cells were thought to be progenitor cells for cardiomyocyte proliferation after specific stimuli. However, the true origin of the proliferating myocardiac cells and the role of Isl1 in adult mammals remain unresolved. In this study, Isl1-CreERT2 knock-in mouse model was constructed using CRISPR/Cas9 technology. Using tamoxifen-inducible Isl1-CreERT/Rosa26R-LacZ system, Isl1+cells and their progeny were permanently marked by lacZ-expression. X-gal staining, immunostaining, and quantitative PCR were then used to reveal the fate of Isl1+cells under physiological and exercise conditions in mouse hearts from embryonic stage to adulthood. Isl1+cells were found to localize to the sinoatrial node, atrioventricular node, cardiac ganglia, aortic arch, and pulmonary roots in adult mice heart. However, they did not act as cardiac progenitor cells under physiological and exercise conditions. Although Isl1+cells showed progenitor cell properties in early mouse embryos (E7.5), this ability was lost by E9.5. Furthermore, although the proliferation and regeneration of heart cell was observed in response to exercise, the cells associated were not Isl1 positive.


Assuntos
Coração/fisiologia , Proteínas com Homeodomínio LIM/genética , Miocárdio/citologia , Miócitos Cardíacos/citologia , Condicionamento Físico Animal , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Proliferação de Células/fisiologia , Mapeamento Cromossômico , Coração/embriologia , Coração/crescimento & desenvolvimento , Masculino , Camundongos Endogâmicos C57BL
15.
Cell Prolif ; 52(6): e12707, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31617266

RESUMO

OBJECTIVES: Fibrosis is a complex process involved in multiple diseases that result in organ injury and failure. Cataract, one common form of ocular fibrosis, is a main cause of blindness worldwide, and surgery may be the only cure. In this regard, epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is the primary cause of anterior subcapsular cataract (ASC). This study aimed to investigate the mechanism by which 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) regulates the function of EMT in LECs. MATERIALS AND METHODS: A mouse model of ASC was used to observe the expression of CNPase in the lens and correlate its expression changes with lens EMT. Furthermore, the effects of CNPase on cell migration and cell proliferation were evaluated by transwell migration, wound healing and EdU staining assays. Finally, Western blotting and immunofluorescence were used to assess the mechanical properties potentially involved in the regulation of EMT by CNPase. RESULTS: The expression of CNPase was upregulated in LECs during the EMT process in mice with ASC. Notably, CNPase significantly promoted the proliferation, migration and EMT of LECs in vitro. Interestingly, the EMT-promoting mechanism of CNPase may be achieved by targeting the Notch signalling pathway. CONCLUSIONS: Considering the involvement of EMT in ASC, both CNPase and the Notch signalling pathway may be therapeutic targets for the treatment of cataracts.


Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Cristalino/metabolismo , Animais , Proliferação de Células/fisiologia , Humanos , Masculino , Camundongos , Receptores Notch/metabolismo , Regulação para Cima
16.
Zhonghua Zhong Liu Za Zhi ; 41(10): 742-747, 2019 Oct 23.
Artigo em Chinês | MEDLINE | ID: mdl-31648495

RESUMO

Objective: To investigate the effect of thymopoietin (TMPO) gene deleted by small interfering RNA (RNAi) on the proliferation and apoptosis of lung cancer cell A549 and its mechanism. Methods: TMPO siRNA was transfected into A549 cells by lipofection. The transfected siRNA control was used as a negative control, and the parent cells were used as blank control. Forty-eight hours later, the expression of TMPO in the transfected cells was detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay, cell cycle and apoptosis were detected by flow cytometry, the protein levels of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, notch receptor 1 (Notch1) and mammalian rapamycin target protein (mTOR) were detected by Western blot analysis. Results: The results of RT-PCR and Western blot showed that the expression levels of TMPO mRNA in the blank control group, the negative control group and TMPO siRNA transfected group were (1.01±0.11), (0.99±0.10), (0.36±0.04), respectively, the protein levels were (0.27±0.02), (0.29±0.03), (0.08±0.10), respectively. The expression levels of TMPO mRNA and protein in the transfected group were significantly lower than those in the blank control and negative control group (P<0.05). The results of MTT assay showed that the OD values of the blank control group, the negative control group and the transfected group were (0.35±0.04), (0.37±0.04) and (0.34±0.03) at 24 h of transfection, respectively. The OD values at 48 h were (0.47±0.06), (0.46±0.08), (0.37±0.04), the OD values at 72 h were (0.75±0.08), (0.73±0.07), (0.49±0.05), respectively, and the OD values at 96 h were (1.09±0.07), (1.06±0.08), (0.56±0.06). The proliferation abilities of the transfected cells at 48, 72, 96 h were significantly lower than those of the blank control and the negative control group (P<0.05). The results of flow cytometry showed that the proportion of G(0)/G(1) phase cells in blank control group, negative control group and transfection group were (62.55±2.03)%, (61.24±3.15)%, (47.35±2.44)%, respectively. The proportion of cells in S phase were (17.12±1.31)%, (17.70±2.01)%, and (20.81±2.06)%, respectively. The proportion of cells in G(2)/M phase were (20.33±1.43)%, (21.06±1.52)%, (31.84±2.76)%, respectively. The proportion of cells in G(0)/G(1) phase of transfection group was significantly lower than those of blank control and negative control group (P<0.05). The proportion of cells in G(2)/M phase of transfection group was significantly higher than those of blank control and negative control group (P<0.05). The apoptosis ratio of the transfection group was (34.10±2.69)%, significantly higher than (2.96±0.03)% of the blank control and (3.01±0.04)% of the negative control group (P<0.05). Western blot analysis showed that PCNA, Notch1 and mTOR proteins were down-regulated while cleaved caspase-3 protein was up-regulated in A549 cells after deletion of TMPO. Conclusion: The inhibition of TMPO gene expression induced by small interfering RNA can significantly inhibit the proliferation and induce apoptosis of A549 cells, and the mechanism is associated with the inhibition of the activation of Notch1/mTOR signaling pathway.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Pulmonares/patologia , Timopoietinas/metabolismo , Animais , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Timopoietinas/genética , Transfecção
17.
Braz Oral Res ; 33: e059, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31664357

RESUMO

We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.


Assuntos
Indutores da Angiogênese/farmacologia , Polpa Dentária/citologia , Polpa Dentária/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adolescente , Adulto , Western Blotting , Contagem de Células , Ensaios de Migração Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno , Polpa Dentária/fisiologia , Combinação de Medicamentos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Laminina , Neovascularização Fisiológica/fisiologia , Proteoglicanas , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Reprodutibilidade dos Testes , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Adulto Jovem
18.
Br J Radiol ; 92(1104): 20190672, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31603350

RESUMO

OBJECTIVE: This paper considers aspects of radiobiology and cell and tissue kinetics applicable to legal disputations concerned with diagnostic and treatment onset delays. METHODS: Various models for tumour volume changes with time are reviewed for estimating volume ranges at earlier times, using ranges of kinetic parameters. Statistical cure probability methods, using Poisson statistics with allowances for parameter heterogeneity, are also described to estimate the significance of treatment delays, as well as biological effective dose (BED) estimations of radiation effectiveness. RESULTS: The use of growth curves, based on parameters in the literature but with extended ranges, can identify a window of earlier times when such tumour volumes would be amenable to a cure based on the literature for curability with stage (and dimensions). Also, where tumour dimensions are not available in a post-operative setting, higher cure probabilities can be achieved if treatment had been given at earlier times. CONCLUSION: The use of radiobiological modelling can provide useful insights, with quantitative assessments of probable prior conditions and future outcomes, and thus be of assistance to a Court in deciding the most correct judgement. ADVANCES IN KNOWLEDGE: This study collates prior knowledge about aspects of radiobiology that can be useful in the accumulation of sufficient proof within medicolegal claims involving diagnostic and treatment days.


Assuntos
Diagnóstico Tardio/legislação & jurisprudência , Neoplasias/diagnóstico , Neoplasias/radioterapia , Radiobiologia/legislação & jurisprudência , Tempo para o Tratamento/legislação & jurisprudência , Algoritmos , Biomarcadores Tumorais/análise , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Estadiamento de Neoplasias , Neoplasias/patologia , Distribuição de Poisson , Resolução de Problemas , Prognóstico , Radioterapia (Especialidade)/legislação & jurisprudência , Radioterapia (Especialidade)/métodos , Radiobiologia/métodos , Eficiência Biológica Relativa , Fatores de Tempo , Carga Tumoral/fisiologia
19.
Cell Prolif ; 52(6): e12703, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31621133

RESUMO

OBJECTIVES: Interleukin-34 (IL-34) is associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). However, the role and associated mechanisms of IL-34 in HBV-related HCC remain unclear. In this study, the expression, biological function and associated mechanisms of IL-34 in HBV-related HCC cells were investigated. METHODS: IL-34 expression induced by HBV and HBV X (HBX) gene was measured in hepatoma cells. The role of CCAAT/enhancer-binding protein α (CEBP/α) in HBX-induced IL-34 expression was examined. The signal pathways involved in the expression of CEBP/α and IL-34 induced by HBX were assessed. The role of IL-34 in the proliferation and migration of HCC cells, and related mechanisms were explored. RESULTS: Dependent on HBX, HBV increased IL-34 expression in hepatoma cells, and HBX upregulated and interacted with CEBP/α to enhance the activity of IL-34 promoters. CEBP/α mediated by HBX was associated with the activation of PI3-K and NF-κB pathways to promote IL-34 expression. Via CSF1-R and CD138, IL-34 promoted the proliferation and migration of hepatoma cells, and contributed to the activation of ERK and STAT3 pathways and the upregulation of Bcl-xl and c-Myc mediated by HBX. CONCLUSION: We demonstrate that IL-34 contributes to HBX-mediated functional abnormality of HCC cells and provides a novel insight into the molecular mechanism of carcinogenesis mediated by HBX.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Interleucinas/metabolismo , Transativadores/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Hepatite B , Humanos , Neoplasias Hepáticas/genética
20.
Anticancer Res ; 39(10): 5427-5436, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570437

RESUMO

BACKGROUND/AIM: Renal cell carcinoma (RCC) is one of the most common tumor diseases in adults, and new specific biomarkers are urgently needed to define diagnosis and prognosis of patients with RCC as well as monitor the outcome of therapeutic interventions. The enzyme nicotinamide N-methyltransferase (NNMT) is believed to represent such a marker molecule in RCC therapy. MATERIALS AND METHODS: NNMT expression was examined by western blotting in samples from patients with RCC and in RCC cell lines. Effects of NNMT on cell growth and metabolism were assessed using the Hoechst 33342 reagent assay and Vita-Orange cell viability assay. Incubation experiments were performed to study the influence of methionine and interleukin-6 (IL6) on expression of NNMT. RESULTS: In patient samples, NNMT was up-regulated depending on the stage of progression. Investigations in an RCC cell culture model showed that after modulation of NNMT expression, cellular metabolism, but not cell growth was affected. This regulatory function was also dependent on the presence of the NNMT precursor substrate methionine and IL6. CONCLUSION: The metabolism-regulatory activity of NNMT depends on the precursor substrate methionine and the presence of IL6. The function of methionine appears to be dependent on the stage of progression, since in individual RCC cell lines, opposing effects on metabolism were demonstrated. This, in turn, reflects the thoroughly complex situation in the clinic.


Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Metionina/metabolismo , Nicotinamida N-Metiltransferase/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Progressão da Doença , Células HEK293 , Humanos , Interleucina-6/metabolismo , Prognóstico , Regulação para Cima/fisiologia
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