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1.
J Gene Med ; 26(10): e3743, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39376029

RESUMO

BACKGROUND: Non-small cell lung cancer (NSCLC) is the main type of lung cancer with high morbidity and mortality. Vascular mimicry (VM), a distinct microcirculation model in tumors that differs from classical angiogenesis, is strongly associated with poor clinical outcomes in cancer patients. miR-491-5p has been reported to prevent NSCLC progression, including proliferation, metastasis, and angiogenesis. However, the effect and mechanism of miR-491-5p on VM have not been studied in NSCLC. METHODS: The expression of miR-491-5p was detected by quantitative reverse transcription PCR (qPCR) and fluorescence in situ hybridization (FISH). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining assays were used to examine cell growth. Tube formation assay was used to assess VM in NSCLC cells. Immunohistochemistry (IHC) and western blot were performed to detect protein expression. Immunoprecipitation was used to confirm the interaction between OTU deubiquitinase 7B (OTUD7B) and vascular endothelial growth factor A (VEGFA), and the level of ubiquitinated VEGFA. A nude mouse tumorigenesis model was used to evaluate the carcinogenic capacity of NSCLC cells in vivo. Luciferase reporter assay was used to identify the potential target of miR-491-5p. RESULTS: MiR-491-5p was found downregulated in NSCLC tissues, and miR-491-5p deficiency was strongly associated with angiogenesis. miR-491-5p mimics suppressed cell viability, migration, and VM. Conversely, an inhibitor of miR-491-5p had the opposite effect. OTUD7B, a deubiquitinase, was identified as a downstream target of miR-491-5p. A luciferase reporter assay indicated that miR-491-5p directly binds to the 3'UTR of OTUD7B. Moreover, mimics of miR-491-5p caused a significant reduction in the OTUD7B protein in NSCLC cells, and an inhibitor of miR-491-5p stabilized the OTUD7B protein. In addition, overexpression of OTUD7B promoted cell proliferation, migration, and VM, similar to the effects of an inhibitor of miR-491-5p. Further exploration revealed that OTUD7B interacts with VEGFA and that the miR-491-5p-OTUD7B axis modulates the ubiquitination of VEGFA. The rescue experiment indicated that OTUD7B compromised the inhibitory effects of miR-491-5p on the cellular function of NSCLC cells. CONCLUSIONS: Overall, our study first proved that miR-491-5p impedes VM by suppressing OUTD7B and promoting the ubiquitination of VEGFA. The miR-491-5p/OTUD7B axis may be a novel target for antiangiogenic therapy in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , MicroRNAs , Neovascularização Patológica , Ubiquitinação , Fator A de Crescimento do Endotélio Vascular , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Animais , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/genética , Movimento Celular/genética , Camundongos Nus , Endopeptidases
2.
Discov Oncol ; 15(1): 247, 2024 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-38935308

RESUMO

Prostate cancer (PCa) is the second leading disease of cancer-related death in men around the world, and it is almost impossible to treat advanced PCa. OTUD7B is a member of the deubiquitinase family that undergoes a post-translational transformation process, which is essential for cell stability and signaling and is known to play a critical role in cancer. However, its role in PCa has not been discovered. The aim of the study was to investigate the expression and mechanism of OTUD7B in PCa cells. According to the database, high OTUD7B expression showed a poor prognosis. Therefore, we downregulated OTUD7B using siRNA and confirmed the role of OTUD7B in PC3 prostate cancer cells. OTUD7B knockdown effectively induced apoptosis and inhibited the proliferation in PC3 cells. OTUD7B knockdown inhibited autophagy through AKT/mTOR signaling. We also confirmed the relationship between AKT/mTOR signaling and autophagy through rapamycin, an mTOR inhibitor. Taken together, OTUD7B promotes the proliferation, and autophagy, and inhibits apoptosis of prostate cancer cells via the AKT/mTOR signaling pathway.

3.
Oncol Lett ; 27(3): 102, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38298430

RESUMO

Breast cancer is a leading cause of cancer-related death in women worldwide; therefore, there is an urgent need to develop novel therapies and drugs that prolong the survival and improve the quality of life of patients with breast cancer. In the present study, the effects and underlying mechanisms of OTU domain-containing 7B (OTUD7B) knockdown on breast cancer were investigated using MDA-MB-468, MDA-MB-453 and MCF7 cell lines. The results of Cell Counting Kit 8, colony formation and tumor sphere formation experiments showed that OTUD7B knockdown caused a significant decrease in the proliferation and sphere formation ability of MDA-MB-468, MDA-MB-453 and MCF7 cells in vitro. Moreover, western blotting results showed that CD44, EpCAM, SOX2 and Nanog protein levels were significantly decreased following OTUD7B knockdown. These findings indicated that OTUD7B knockdown reduced the proliferation and stemness of breast cancer cells. Co-immunoprecipitation assays demonstrated that OTUD7B interacted with forkhead box protein M1 (FOXM1) and reduced the polyubiquitylation of FOXM1 in breast cancer cells; accordingly, FOXM1 protein levels were significantly decreased by OTUD7B knockdown. Furthermore, the overexpression of FOXM1 reduced the inhibitory effects of OTUD7B knockdown on breast cancer cells. The findings of the present study provide new insights into the oncogenic role of OTUD7B in breast cancer and indicate that OTUD7B may serve as a therapeutic target for breast cancer.

4.
Dig Liver Dis ; 56(2): 352-362, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37429790

RESUMO

BACKGROUND: Gastric cancer (GC) is one of the most common malignancies worldwide. Ovarian tumor protein superfamily serves a crucial role in tumor growth progression, among them, ovarian tumor domain-containing 7B (OTUD7B) as a deubiquitinase (DUB) is frequently found in various cancers, but the role of OTUD7B in GC is poorly understood. AIMS: To clarify the effect of OTUD7B on GC progression. METHODS: Functional experiments were performed to detect the proliferation, migration and invasion of GC cells. Xenografts were used to measure the effects in vivo. Co-immunoprecipitation (Co-IP) and ubiquitination assays showed the interaction of OTUD7B and YAP1. RESULTS: OTUD7B was highly expressed in tumor tissues from GC patients, and high mRNA expression was strongly associated with poor prognosis, suggesting that OTUD7B was an independent prognostic factor. Moreover, OTUD7B overexpression promoted GC cell proliferation and metastasis both in vitro and in vivo, whereas OTUD7B knockdown exhibited opposing biological effects. Mechanically, OTUD7B promoted downstream target genes of YAP1 including NUAK2, Snail, Slug, CDK6, CTGF, and BIRC5. Importantly, OTUD7B enhanced the activation of YAP1 via deubiquitinating and stabilizing to upregulate NUAK2 expression. CONCLUSIONS: OTUD7B is a novel DUB of the YAP1 pathway and accelerates GC progression. Therefore, OTUD7B may be a promising therapeutic target against GC.


Assuntos
Neoplasias Ovarianas , Neoplasias Gástricas , Feminino , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Neoplasias Gástricas/patologia , Fatores de Transcrição/genética , Proteínas de Sinalização YAP
5.
Environ Toxicol ; 39(4): 1989-2005, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38088504

RESUMO

Hyperthermia, as an adjuvant therapy, has shown promising anti-tumor effects. Ovarian tumor domain-containing 7B (OTUD7B) is a deubiquitinating enzyme that is frequently found in a variety of cancers. The aim of this study is to investigate the role of OTUD7B in lung cancer hyperthermia and the underlying mechanism. A549 and CALU-3 cells were respectively exposed to 42 or 44°C for the indicated times (0, 1, 3, or 6 h) followed by incubation at 37°C for 24 h. We found a temperature- and time-dependent decrease in cell viability and an increase in apoptosis levels. Compared with 0 h, heat treatment for 3 h inhibited the proliferation and invasion of A549 cells, reduced the expression levels of mitochondrial membrane potential, IAP family members (cIAP-1 and XIAP) proteins and ubiquitination of Smac, and increased Smac protein expression. Treatment with 10 µM Smac mimic BV6 further enhanced the anti-tumor effect of hyperthermia. Next, co-IP validation showed that OTUD7B interacted with Smac and stabilized Smac through deubiquitination. OTUD7B overexpression induced damage in A549 and CALU-3 cells, while silencing OTUD7B caused opposite effects. Overexpressing OTUD7B enhanced the anti-cancer effect of hyperthermia, while si-OTUD7B reversed the anti-cancer effect of hyperthermia, which was verified in the xenograft tumor model in nude mice. Taken together, OTUD7B may serve as a potential anticancer factor with potential clinical efficacy in the thermotherapeutic treatment of lung cancer.


Assuntos
Hipertermia Induzida , Neoplasias Pulmonares , Doenças Mitocondriais , Animais , Humanos , Camundongos , Apoptose , Linhagem Celular Tumoral , Enzimas Desubiquitinantes , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos Nus , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/farmacologia
6.
Biomolecules ; 13(6)2023 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-37371581

RESUMO

The Wnt signaling pathway plays a critical role in regulating normal cellular processes, including proliferation, differentiation, and apoptosis. Dysregulation of Wnt signaling has been implicated in various human diseases, including cancer. ß-catenin and LEF1 are key mediators of Wnt signaling, and their dysregulation is a hallmark of many cancer types. In this study, we aimed to identify the deubiquitinases (DUBs) that regulate the Wnt signaling pathway through the essential component LEF1. Screening candidate DUBs from the human DUB library, we discovered that OTUD7B interacts with LEF1 and activates Wnt signaling. OTUD7B and LEF1 interact with each other through the UBA and HMG domains, respectively. Furthermore, OTUD7B promotes the nuclear localization of LEF1, leading to an increased interaction with ß-catenin in the nucleus while not noticeably affecting ubiquitination on LEF1. Using qPCR array analysis, we found that OTUD7B overexpression leads to an upregulation of 75% of the tested Wnt target genes compared to the control. These findings suggest that OTUD7B may serve as a potential therapeutic target in human diseases, including cancers where Wnt signaling is frequently dysregulated.


Assuntos
Neoplasias , Via de Sinalização Wnt , Humanos , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Ativação Transcricional , Regulação para Cima , Endopeptidases , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo
7.
Cancers (Basel) ; 15(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36672466

RESUMO

Protein deubiquitinases play critical pathophysiological roles in cancer. Among all deubiquitinases, an oncogenic function for OTUD7B has been established in genetic NSCLC murine models. However, few deubiquitinase inhibitors have been developed due to technical challenges. Here, we report a putative small molecule OTUD7B inhibitor obtained from an AI-aided screen of a 4 million compound library. We validated the effects of the OTUD7B inhibitor (7Bi) in reducing Akt-pS473 signals in multiple NSCLC and HEK293 cells by blocking OTUD7B-governed GßL deubiquitination in cells, as well as inhibiting OTUD7B-mediated cleavage of K11-linked di-ub in an in vitro enzyme assay. Furthermore, we report in leukemia cells, either genetic depletion or 7Bi-mediated pharmacological inhibition of OTUD7B reduces Akt-pS473 via inhibiting the OTUD7B/GßL signaling axis. Together, our study identifies the first putative OTUD7B inhibitor showing activities both in cells and in vitro, with promising applications as a therapeutic agent in treating cancer with OTUD7B overexpression.

8.
Arch Biochem Biophys ; 724: 109266, 2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35523269

RESUMO

Fibrosis is one of the crucial reasons for cardiac dysfunction after myocardial infarction (MI). Understanding the underlying molecular mechanism that causes fibrosis is crucial to developing effective therapy. Recently, OUT domain-containing 7B (OTUD7B), also called Cezanne, a multifunctional deubiquitylate, has been found to play various roles in cancer and vascular diseases and control many important signaling pathways, including inflammation, proliferation, and so on. However, whether OTUD7B plays a role in fibrosis caused by MI remains unclear. Our study aimed to explore the function of OTUD7B in cardiac fibrosis and investigate the underlying mechanism. We found that the expression of OTUD7B was downregulated in the MI rat model and cultured cardiac fibroblasts (CFs) in hypoxic conditions and after TGF-ß1 treatment. In vitro, silencing OTUD7B using small interfering RNA (siRNA) increased α-SMA (smooth muscle actin α) and collagen Ⅰ levels in CFs, whereas the overexpression of OTUD7B using adenovirus decreased their expression. Mechanistically, OTUD7B could regulate the phosphorylation of focal adhesion kinase (FAK), a non-receptor tyrosine kinase that has been proved to act as a potential mediator of fibrosis, and ERK/P38 MAPK was involved in this regulation process. In vitro, overexpression of OTUD7B downregulated the phosphorylation level of FAK and then inhibited ERK/P38 phosphorylation, thus leading to decreased α-SMA and collagen Ⅰ expressions, while OTUD7B knockdown showed an opposite result. These findings suggest that OTUD7B could become a potentially effective therapeutic strategy against fibrosis after MI.


Assuntos
Endopeptidases/metabolismo , Infarto do Miocárdio , Animais , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibrose , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
J Cancer ; 13(3): 998-1004, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154465

RESUMO

OTUD7B is a deubiquitinase and has been reported as a prognostic factor in various solid tumors. However, its prognostic value in lymphoma patients remains unclear. We detected OTUD7B expression levels in 160 diffuse large B-cell lymphoma (DLBCL) tissue samples by immunohistochemistry, and analyzed correlations between its expression and clinic-pathologic parameters as well as clinical outcomes. We also investigated association between OTUD7B expression and chemotherapeutic drugs anti-tumor activity in vitro. We found that OTUD7B overexpressed in 129 (80.6%) cases, and patients with overexpression of OTUD7B experienced better overall survival comparing to those with OTUD7B low expression (P=0.021). Multivariate Cox regression analysis illustrated that OTUD7B was an independent prognostic indicator. In DLBCL cell lines, we found that Chidamide could up-regulate OTUD7B in several DLBCL cell lines, and also had synergistic effect with doxorubicin at low concentration. Our data illustrated that OTUD7B deficiency is a negative predictor of clinical outcome, and might be a potential therapeutic target in the treatment of diffuse large B-cell lymphoma.

10.
Autophagy ; 17(6): 1367-1378, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-32453962

RESUMO

Macroautophagy/autophagy, a eukaryotic homeostatic process that sequesters cytoplasmic constituents for lysosomal degradation, is orchestrated by a number of autophagy-related (ATG) proteins tightly controlled by post-translational modifications. However, the involvement of reversible ubiquitination in the regulation of autophagy remains largely unclear. Here, we performed a single-guide RNA-based screening assay to investigate the functions of deubiquitinating enzymes (DUBs) in regulating autophagy. We identified previously unrecognized roles of several DUBs in modulating autophagy at multiple levels by targeting various ATG proteins. Mechanistically, we demonstrated that STAMBP/AMSH (STAM-binding protein) promotes the stabilization of ULK1 by removing its lysine 48 (K48)-linked ubiquitination, whereas OTUD7B mediates the degradation of PIK3 C3 by enhancing its K48-linked ubiquitination, thus positively or negatively affects autophagy flux, respectively. Together, our study elaborated on the broad involvement of DUBs in regulating autophagy and uncovered the critical roles of the reversible ubiquitination in the modification of ATG proteins.Abbreviations: ATG: autophagy-related; Baf A1: bafilomycin A1; DUB: deubiquitinating enzyme; EBSS: Earle's balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OTUD7B: OTU domain-containing protein 7B; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; sgRNA: single-guide RNA; SQSTM1/p62: sequestosome 1; STAMBP/AMSH: STAM-binding protein; ULK1: unc-51 like autophagy activating kinase 1; USP: ubiquitin specific peptidase.


Assuntos
Autofagia/fisiologia , Ensaios de Triagem em Larga Escala , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Endopeptidases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Lisossomos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia
11.
J Exp Clin Cancer Res ; 39(1): 244, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198776

RESUMO

BACKGROUND: Smac mimetics are a type of drug that can induce apoptosis by antagonizing IAP family members in cancer treatment. However, a recent study showed that Smac mimetics can trigger cell invasion and migration in cancer cells by activating the NF-κB pathway. METHODS: We assessed lung cancer cell elongation, invasion and migration under treatment with the Smac mimetic LCL161. Functional analyses (in vitro and in vivo) were performed to detect the contribution of NIK and OTUD7B to LCL161-induced cell invasion and migration. The role of OTUD7B in regulation of the TRAF3/NIK/NF-κB pathway under LCL161 treatment was analysed by immunoblotting, immunoprecipitation, luciferase and ubiquitin assays, shRNA silencing and plasmid overexpression. Expression levels of OTUD7B, NIK and TRAF3 in tissue samples from lung cancer patients were examined by immunohistochemistry. RESULTS: We found that LCL161 stimulates lung cancer cell elongation, invasion and migration at non-toxic concentrations. Mechanistically, LCL161 results in NIK accumulation and activates the non-canonical rather than the canonical NF-κB pathway to enhance the transcription of target genes, such as IL-2 and MMP-9. Importantly, knockdown of NIK dramatically suppresses LCL161-induced cell invasion and migration by reducing the proteolytic processing of p100 to p52 and target gene transcription. Interestingly, we discovered that OTUD7B increases TRAF3 and decreases NIK to inhibit the non-canonical NF-κB pathway and that overexpression of OTUD7B suppresses LCL161-induced cell invasion and migration. Notably, OTUD7B directly binds to TRAF3 rather than to NIK and deubiquitinates TRAF3, thereby inhibiting TRAF3 proteolysis and preventing NIK accumulation and NF-κB pathway activation. Furthermore, the OTU domain of OTUD7B is required for the inhibition of LCL161-induced cell invasion and migration, as demonstrated by transfection of the C194S/H358R(CH) mutant OTUD7B. Finally, we investigated whether OTUD7B inhibits LCL161-induced lung cancer cell intrapulmonary metastasis in vivo, and our analysis of clinical samples was consistent with the above findings. CONCLUSIONS: Our study highlights the importance of OTUD7B in the suppression of LCL161-induced lung cancer cell invasion and migration, and the results are meaningful for selecting lung cancer patients suitable for LCL161 treatment.


Assuntos
Endopeptidases/toxicidade , Neoplasias Pulmonares/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Endopeptidases/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , Análise de Sobrevida
12.
J Biol Rhythms ; 35(6): 542-554, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32748687

RESUMO

Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) play critical roles in the epigenetic and transcriptional regulation of mammalian circadian systems. Circadian rhythmicity regulates many aspects of our immune system, and perturbation of the circadian clock can augment the inflammatory response. However, knowledge of the precise functions of lncRNAs in the regulation of immune functions within the circadian system is relatively limited. In this study, differentially expressed lncRNAs induced by Clock knockdown were screened via mRNA/lncRNA microarray and bioinformatic prediction analysis. We identified a Clock-regulated lncRNA, AK028245, which was correlated with the activation of the immune response. The expression levels of AK028245 were decreased in the spleen of immunosuppressed mice and elevated in immune-activated mice treated with lipopolysaccharide (LPS). Further, Clock knockdown decreased the expression of OTUD7B and A20, 2 early immune response factors acting on the NF-κB signaling pathway. Interestingly, inhibition of AK028245 increased their expression, mitigating the effects of Clock knockdown. In addition, inhibition of AK028245 downregulated the expression of tumor necrosis factor-α and interleukin-6 in the late stages of LPS stimulation and the expression of interferon-γ and Cxcl12 in the peak stages. We conclude that this newly identified lncRNA plays a role in the crosstalk between Clock and immune response regulators, likely resulting in a proinflammatory response targeting OTUD7B and A20. The lncRNA AK028245 has revealed a new mechanism of the immune response and provided new targets for the treatment of immune disorders.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Endopeptidases/imunologia , Imunidade/genética , RNA Longo não Codificante/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Animais , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro
13.
Theranostics ; 10(17): 7787-7811, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32685020

RESUMO

Objective: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are essential for vascular remodeling. Natural compounds with diterpene chinone or phenolic acid structure from Salvia miltiorrhiza, an eminent medicinal herb widely used to treat cardiovascular diseases in China, can effectively attenuate vascular remodeling induced by vascular injury. However, it remains unknown whether Salvia miltiorrhiza-derived miRNAs can protect VSMCs from injury by environmental stimuli. Here, we explored the role and underlying mechanisms of Salvia miltiorrhiza-derived Sal-miR-1 and 3 in the regulation of VSMC migration and monocyte adhesion to VSMCs induced by thrombin. Methods: A mouse model for intimal hyperplasia was established by the ligation of carotid artery and the injured carotid arteries were in situ-transfected with Sal-miR-1 and 3 using F-127 pluronic gel. The vascular protective effects of Sal-miR-1 and 3 were assessed via analysis of intimal hyperplasia with pathological morphology. VSMC migration and adhesion were analyzed by the wound healing, transwell membrane assays, and time-lapse imaging experiment. Using loss- and gain-of-function approaches, Sal-miR-1 and 3 regulation of OTUD7B/KLF4/NMHC IIA axis was investigated by using luciferase assay, co-immunoprecipitation, chromatin immunoprecipitation, western blotting, etc. Results:Salvia miltiorrhiza-derived Sal-miR-1 and 3 can enter the mouse body after intragastric administration, and significantly suppress intimal hyperplasia induced by carotid artery ligation. In cultured VSMCs, these two miRNAs inhibit thrombin-induced the migration of VSMCs and monocyte adhesion to VSMCs. Mechanistically, Sal-miR-1 and 3 abrogate OTUD7B upregulation by thrombin via binding to the different sites of the OTUD7B 3'UTR. Most importantly, OTUD7B downregulation by Sal-miR-1 and 3 attenuates KLF4 protein levels via decreasing its deubiquitylation, whereas decreased KLF4 relieves its repression of transcription of NMHC IIA gene and thus increases NMHC IIA expression levels. Further, increased NMHC IIA represses VSMC migration and monocyte adhesion to VSMCs via maintaining the contractile phenotype of VSMCs. Conclusions: Our studies not only found the novel bioactive components from Salvia miltiorrhiza but also clarified the molecular mechanism underlying Sal-miR-1 and 3 inhibition of VSMC migration and monocyte adhesion to VSMCs. These results add important knowledge to the pharmacological actions and bioactive components of Salvia miltiorrhiza. Sal-miR-1 and 3-regulated OTUD7B/KLF4/NMHC IIA axis may represent a therapeutic target for vascular remodeling.


Assuntos
MicroRNAs/farmacologia , RNA de Plantas/farmacologia , Salvia miltiorrhiza/genética , Túnica Íntima/patologia , Remodelação Vascular/efeitos dos fármacos , Animais , Artérias Carótidas/citologia , Artérias Carótidas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Endopeptidases/metabolismo , Humanos , Hiperplasia/tratamento farmacológico , Hiperplasia/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , MicroRNAs/uso terapêutico , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Cadeias Pesadas de Miosina/metabolismo , RNA de Plantas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Túnica Íntima/efeitos dos fármacos
14.
Curr Eye Res ; 45(12): 1514-1525, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32438838

RESUMO

PURPOSE: Choroidal neovascularization (CNV) is the key pathological change caused by irreversible blindness resulting from neovascular AMD (nAMD). However, the pathological mechanisms underlying CNV remain largely unknown. Here, we aimed to investigate the role of miR-146a-5p in CNV formation. MATERIALS AND METHODS: At the cellular level, we overexpressed or downregulated miR-146a-5p in an umbilical vein endothelial cell line (EA.hy926) by transfecting cells with either a miR-146a-5p mimic or an inhibitor. CCK8, wound healing, and Matrigel assays were performed to examine the proliferation, migration, and tube formation of endothelial cells (EA.hy926). Target relationship between miR-146a-5p and OTUD7B was verified using a double luciferase reporter experiment. An experimental CNV model was established by treating fundi of male C57BL/6 J mice with 810 nm laser. Fundus fluorescein angiography (FFA) was performed to evaluate the leakage of CNV on day 7 after miR-146a-5p antagomir intravitreal injection. The CNV volume was measured using Choroidal Flatmounts in a confocal study. The expression levels of VEGF, ICAM1, and NF-κB (p50 and p65) were detected both in vitro and in vivo. RESULTS: The expression of miR-146a-5p was increased in LPS-stimulated endothelial cells and in experimental CNV RPE-choroidal complexes in mouse models. LPS-induced proliferation, migration, and tube formation were inhibited by the miR-146a-5p inhibitor. The miR-146a-5p antagomir attenuated CNV formation and fluorescent leakage in the vivo CNV model. In the LPS-stimulated endothelial cells and the CNV mouse model, the NF-κB signaling pathway was activated and the expression of VEGF and ICAM1 increased. Conversely, downregulation of miR-146a-5p inactivated the NF-κB signaling pathway and reduced the expression of VEGF and ICAM1. CONCLUSIONS: Our results indicated that downregulation of miR-146a-5p inhibited experimental CNV formation via inactivation of the NF-κB signaling pathway.


Assuntos
Neovascularização de Coroide/prevenção & controle , Endopeptidases/genética , Regulação da Expressão Gênica/fisiologia , MicroRNAs/genética , NF-kappa B/metabolismo , Animais , Western Blotting , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Regulação para Baixo , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Angiofluoresceinografia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acuidade Visual
15.
Semin Cancer Biol ; 67(Pt 2): 145-158, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32201366

RESUMO

Since its discovery forty years ago, protein ubiquitination has been an ever-expanding field. Virtually all biological processes are controlled by the post-translational conjugation of ubiquitin onto target proteins. In addition, since ubiquitin controls substrate degradation through the action of hundreds of enzymes, many of which represent attractive therapeutic candidates, harnessing the ubiquitin system to reshape proteomes holds great promise for improving disease outcomes. Among the numerous physiological functions controlled by ubiquitin, the cell cycle is among the most critical. Indeed, the discovery that the key drivers of cell cycle progression are regulated by the ubiquitin-proteasome system (UPS) epitomizes the connection between ubiquitin signaling and proliferation. Since cancer is a disease of uncontrolled cell cycle progression and proliferation, targeting the UPS to stop cancer cells from cycling and proliferating holds enormous therapeutic potential. Ubiquitination is reversible, and ubiquitin is removed from substrates by catalytic proteases termed deubiquitinases or DUBs. While ubiquitination is tightly linked to proliferation and cancer, the role of DUBs represents a layer of complexity in this landscape that remains poorly captured. Due to their ability to remodel the proteome by altering protein degradation dynamics, DUBs play an important and underappreciated role in the cell cycle and proliferation of both normal and cancer cells. Moreover, due to their enzymatic protease activity and an open ubiquitin binding pocket, DUBs are likely to be important in the future of cancer treatment, since they are among the most druggable enzymes in the UPS. In this review we summarize new and important findings linking DUBs to cell cycle and proliferation, as well as to the etiology and treatment of cancer. We also highlight new advances in developing pharmacological approaches to attack DUBs for therapeutic benefit.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Neoplasias/patologia , Ciclo Celular , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/metabolismo , Enzimas Desubiquitinantes/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mitose , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo
16.
Inflammation ; 43(3): 975-984, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31940107

RESUMO

The aim of this article is to study the effect of miR-486-5p in acute lung injury (ALI). MiR-486-5p expression in peripheral blood was determined in ALI patients and healthy volunteers by qRT-PCR. ALI mouse model were reproduced by LPS treatment, and miR-486-5p NC and miRNA-486 inhibitors were injected through trachea. ALI patients' peripheral blood and LPS-induced acute lung injury in mice had significantly higher miR-486-5p levels than control subjects. Inhibition of miR-486-5p by injection with antagomiR-486-5p markedly reduced LPS-induced lung inflammation. Moreover, knockdown of miR-486-5p can reduce protects A549 cell against LPS-induced injury and its corresponding inflammatory response. In addition, Mechanistic analysis indicated that miR-486-5p on the occurrence of ALI is related to the inhibition of OTUD7B activity, which induces the downregulation of inflammatory in ALI. Our results identified miR-486-5p independently associated with ALI. miR-486-5p can mediate the formation of ALI by promoting inflammation.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Apoptose/fisiologia , Endopeptidases/metabolismo , MicroRNAs/metabolismo , Células A549 , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Endopeptidases/genética , Técnicas de Inativação de Genes/métodos , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética
17.
J Biol Chem ; 295(8): 2160-2174, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31937588

RESUMO

Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain-containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63-, and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a noncanonical surface and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez Our results indicate that UBACez can indeed fulfill this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.


Assuntos
Asparagina/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Oxigênio/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Sequência Consenso , Células HEK293 , Humanos , Hidroxilação , Oxigenases de Função Mista/metabolismo , Poliubiquitina/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade
18.
J Exp Clin Cancer Res ; 38(1): 470, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747939

RESUMO

BACKGROUND: Accumulation evidence indicates the vital role of long non-coding RNAs (lncRNAs) in tumorigenesis and the progression of malignant tumors, including pancreatic cancer (PC). However, the role and the molecular mechanism of long non-coding RNA 00976 is unclear in pancreatic cancer. METHODS: In situ hybridization (ISH) and qRT-PCR was performed to investigate the association between linc00976 expression and the clinicopathological characteristics and prognosis of patients with PC. Subsequently, linc00976 over-expression vector and shRNAs were transfected into PC cells to up-regulate or down-regulate linc00976 expression. Loss- and gain-of function assays were performed to investigate the role of linc00976 in proliferation and metastasis in vitro and vivo. ITRAQ, bioinformatic analysis and rescue assay were used to illustrate the ceRNA mechanism network of linc00976/miR-137/OTUD7B and its downstream EGFR/MAPK signaling pathway. RESULTS: linc00976 expression was overexpressed in PC tissues and cell lines and was positively associated with poorer survival in patients with PC. Function studies revealed that linc00976 knockdown significantly suppressed cell proliferation, migration and invasion in vivo and in vitro, whereas its overexpression reversed these effects. Based on Itraq results and online database prediction, Ovarian tumor proteases OTUD7B was found as a downstream gene of linc00976, which deubiquitinated EGFR mediates MAPK signaling activation. Furthermore, Bioinformatics analysis and luciferase assays and rescue experiments revealed that linc00976/miR137/OTUD7B established the ceRNA network modulating PC cell proliferation and tumor growth. CONCLUSION: The present study demonstrates that linc00976 enhances the proliferation and invasion ability of PC cells by upregulating OTUD7B expression, which was a target of miR-137. Ultimately, OTUD7B mediates EGFR and MAPK signaling pathway, suggesting that linc00976/miR-137/OTUD7B/EGFR axis may act as a potential biomarker and therapeutic target for PC.


Assuntos
Endopeptidases/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Neoplasias Pancreáticas/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Endopeptidases/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Transfecção
19.
Front Oncol ; 9: 862, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31572671

RESUMO

OTUD7B, a multifunctional deubiquitinylase, plays an essential role in inflammation and proliferation signals. However, its function in lung cancer remains largely unknown. The aim of this study was to evaluate the prognostic significance of OTUD7B in patients with lung adenocarcinoma and squamous carcinoma and to characterize its molecular mechanisms in lung cancer progression and metastasis. Two tissue microarrays containing 150 pairs of lung squamous carcinoma and matched adjacent non-cancer tissues, and one tissue microarray containing 75 pairs of lung adenocarcinoma and adjacent non-cancer tissues were included, and immunohistochemical staining was performed to assess the clinical relevance of OTUD7B in non-small cell lung cancer. OTUD7B is highly expressed in both lung squamous carcinoma and adenocarcinoma and correlates with a worse prognosis. MTT proliferation, colony formation, migration and invasion assays and immunoblotting assay in NCI-H358 and A549 cell lines suggested that OTUD7B enhances EGF-induced Akt signal transduction and promotes lung cancer cell proliferation and migration. Immunohistochemical staining of large-scale lung cancer subjects (171 cases) revealed positive correlation of OTUD7B and VEGF expression. ELISA and tube formation assay revealed OTUD7B promotes VEGF production and angiogenesis. NCI-H358 tumor model demonstrated OTUD7B is required for lung tumor progression by facilitating activation of Akt signaling. These findings collectively identified OTUD7B as an independent predictive factor for the prognosis of non-small cell lung cancer and revealed OTUD7B promotes lung cancer cell proliferation and metastasis via Akt/VEGF signal pathway.

20.
Oncotarget ; 9(1): 553-565, 2018 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-29416635

RESUMO

Paclitaxel is a first-line chemotherapeutic for patients with breast cancer, particularly triple-negative breast cancer (TNBC). Molecular markers for predicting pathologic responses to paclitaxel treatment is thus urgently needed since paclitaxel resistance is still a clinical issue in treating TNBCs. We investigated the transcriptional profiling of consensus genes in HCC38 (paclitaxel-sensitive) and MDA-MB436 (paclitaxel-resistant) TNBC cells post-treatment with paclitaxel. We found that OTUD7B was downregulated in HCC38 but upregulated in MDA-MB436 cells after paclitaxel treatment at cytotoxic concentrations. Moreover, our data showed that OTUD7B expression causally correlated with IC50 of paclitaxel in a panel of TNBC cell lines. Moreover, we found that OTUD7B upregulation was significantly detected in primary breast cancer tissues compared to normal breast tissues but inversely correlated with tumor growth in TNBC cells. Besides, the increased levels of OTUD7B transcript appeared to causally associate with invasive potentials in TNBC cells. In assessments of recurrence/metastasis-free survival probability, high-levels of OTUD7B transcripts strongly predicted a poor prognosis and unfavorable response to paclitaxel-based chemotherapy in patients with TNBCs. In silico analysis suggested that OTUD7B regulation, probably owing to miR-1180 downregulation, may negatively regulate the NF-κB-Lin28 axis which in turn triggers Let-7 microRNA-mediated caspase-3 downregulation, thereby conferring paclitaxel resistance in TNBCs. These findings suggest that OTUD7B may be a useful biomarker for predicting the anti-cancer effectiveness of paclitaxel and could serve as a new drug target for enhancing the canceridal efficiency of paclitaxel against TNBCs.

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