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1.
Mil Med Res ; 8(1): 49, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34488908

RESUMO

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses. Herein we investigate their functions in human epithelial cells, the primary and initial target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A deficiency in MDA5, RIG-I or mitochondrial antiviral signaling protein (MAVS) enhanced viral replication. The expression of the type I/III interferon (IFN) during infection was impaired in MDA5-/- and MAVS-/-, but not in RIG-I-/-, when compared to wild type (WT) cells. The mRNA level of full-length angiotensin-converting enzyme 2 (ACE2), the cellular entry receptor for SARS-CoV-2, was ~ 2.5-fold higher in RIG-I-/- than WT cells. These data demonstrate MDA5 as the predominant SARS-CoV-2 sensor, IFN-independent induction of ACE2 and anti-SARS-CoV-2 role of RIG-I in epithelial cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , COVID-19/imunologia , Proteína DEAD-box 58/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Receptores Imunológicos/metabolismo , SARS-CoV-2/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Linhagem Celular , Proteína DEAD-box 58/genética , Humanos , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Interferons/metabolismo , Receptores Imunológicos/genética , Transdução de Sinais , Replicação Viral
2.
Nat Commun ; 12(1): 5263, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489457

RESUMO

Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeo Hidrolases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Evolução Molecular , Células HEK293 , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Lenalidomida/química , Lenalidomida/farmacologia , Camundongos , Organoides , Peptídeo Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445421

RESUMO

The Hedgehog (HH) signaling pathway plays an important role in embryonic development and adult organ homeostasis. Aberrant activity of the Hedgehog signaling pathway induces many developmental disorders and cancers. Recent studies have investigated the relationship of this pathway with various cancers. GPCR-like protein Smoothened (SMO) and the glioma-associated oncogene (GLI1) are the main effectors of Hedgehog signaling. Physalin A, a bioactive substance derived from Physalis alkekengi, inhibits proliferation and migration of breast cancer cells and mammospheres formation. Physalin A-induced apoptosis and growth inhibition of mammospheres, and reduced transcripts of cancer stem cell (CSC) marker genes. Physalin A reduced protein expressions of SMO and GLI1/2. Down-regulation of SMO and GLI1 using siRNA inhibited mammosphere formation. Physalin A reduced mammosphere formation by reducing GLI1 gene expression. Down-regulation of GLI1 reduced CSC marker genes. Physalin A reduced protein level of YAP1. Down-regulation of YAP1 using siRNA inhibited mammosphere formation. Physalin A reduced mammosphere formation through reduction of YAP1 gene expression. Down-regulation of YAP1 reduced CSC marker genes. We showed that treatment of MDA-MB-231 breast cancer cells with GLI1 siRNA induced inhibition of mammosphere formation and down-regulation of YAP1, a Hippo pathway effector. These results show that Hippo signaling is regulated by the Hedgehog signaling pathway. Physalin A also inhibits the canonical Hedgehog and Hippo signaling pathways, CSC-specific genes, and the formation of mammospheres. These findings suggest that physalin A is a potential therapeutic agent for targeting CSCs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Vitanolídeos/farmacologia , Proteína GLI1 em Dedos de Zinco/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
4.
Nat Commun ; 12(1): 4789, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373451

RESUMO

CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.


Assuntos
Neoplasias Pulmonares/genética , Oncogenes/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Técnicas de Inativação de Genes , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Terapia de Alvo Molecular , Mutação , Células NIH 3T3 , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras
5.
Ann Clin Lab Sci ; 51(4): 470-486, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34452885

RESUMO

OBJECTIVE: Epithelium-specific ETS protein 3 (Ese-3) is a member of the ETS family that is associated with tumor progression. However, there is little knowledge about Ese-3 in skin cancer. This study was conducted to explore the effects of Ese-3 on clinical prognosis in skin cancer and the functions of HaCaT cells. MATERIALS AND METHODS: Gene expression and clinical data were collected from The Cancer Genome Atlas (TCGA), The Genotype-Tissue Expression (GTEx), and three GSE datasets (GSE15605, GSE46517, and GSE114445). Comparison of data between groups was performed by Student's t-test and chi square test. Survival analysis was performed using log-rank test. Univariate and multivariate analyses were performed using Cox proportional hazards models. Enrichment analysis was used to predict Ese-3 related functions. Cell proliferation assays, colony formation assays, and flow cytometry were used to assess cell proliferation, while Transwell assays analyzed cell migration and invasion. RESULTS: Compared with normal tissues, the Ese-3 mRNA in cutaneous malignant melanoma (CMM) patients was downregulated (P<0.0001). Ese-3 mRNA was associated with the T stage (χ 2=10.015, P=0.018), clinical stage (χ 2=4.122, P=0.042), and prognosis in CMM patients (P=0.0219) and was an independent prognostic predictor in CMM (HR=1.878, P=0.048). Enrichment analysis showed that differentially expressed proteins were associated with "protein kinase B (AKT) binding." CONCLUSION: Ese-3 inhibited the proliferation, migration, and invasion of HaCaT cells by downregulating PSIP1 and NUCKS1 expression levels to inactivate the phosphorylation of AKT.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Neoplasias Cutâneas/patologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Feminino , Células HaCaT , Humanos , Masculino , Invasividade Neoplásica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prognóstico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Taxa de Sobrevida , Fatores de Transcrição/genética
6.
Ann Clin Lab Sci ; 51(4): 487-493, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34452886

RESUMO

OBJECTIVE: Hepatitis B X-interacting protein (HBXIP) interacts with hepatitis B virus X protein to participate in the replication of the hepatitis B virus and carcinogenesis. Cellular growth and metastasis of non-small-cell lung cancer (NSCLC) are repressed by HBXIP inhibition. However, the role and mechanism of HBXIP on NSCLC cell growth remain unknown. MATERIALS: Expression of HBXIP was assessed by qRT-PCR and Western blot. siRNA targeting HBXIP was applied to detect cell viability and proliferation by MTT and colony formation assays. In vivo tumor growth was assessed, and anti-tumor immunity was determined by flow cytometry. The downstream partners involved in HBXIP-mediated tumorigenesis were detected by Western blot. RESULTS: Expression of HBXIP and neuropilin1-1 (NRP-1) was higher in NSCLC tissues and cells than in paracancerous tissues and human lung epithelial cells. siRNA-mediated knockdown of HBXIP decreased the cell viability of NSCLC and suppressed proliferation. Protein expression of Lin28B and NRP-1 was reduced by the knockdown of HBXIP, and over-expression of Lin28B attenuated the HBXIP silence-induced decrease of NRP-1. In vivo tumor growth was suppressed by HBXIP silencing, and the knockdown of HBXIP enhanced anti-tumor immunity through the increase of CD4+ and CD8+ T lymphocytes. CONCLUSION: Down-regulation of HBXIP reduced Lin28B-mediated NRP-1 to suppress NSCLC cell growth and enhance anti-tumor immunity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Neuropilina-1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neuropilina-1/genética , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Eur J Med Res ; 26(1): 93, 2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34391478

RESUMO

BACKGROUND: To investigate the value of Dickkopf-related protein 3 (DKK3) on aerobic glycolysis in pancreatic cancer cells, where DKK3-overexpression is used to determine its effects on CD4+ T cells. METHODS: The BxPC-3-DKK3 cell line was constructed, and peripheral blood mononuclear cell (PBMC) was prepared. After isolated the CD4+ T cells, the lactic acid, glucose uptake ability, cellular viability, proliferation, apoptosis, and markers were detected by PCR and western blot, and the concentrations of multiple cytokines were determined using the ELISA method. RESULTS: After co-culture with pancreatic cancer cells overexpressing DKK3, the glucose uptake markedly, proliferation enhanced and apoptosis inhibited in CD4+ T cells. The co-culture model also revealed that DKK3-overexpression promotes the activation and regulates the metabolism and function of CD4+ T cells. CONCLUSIONS: DKK3 alters the metabolic microenvironment of pancreatic cancer cells and further facilitates the function of CD4+ T cells which suggesting that DKK3 may have a therapeutic potential in pancreatic cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Linfocitária , Neoplasias Pancreáticas/metabolismo , Efeito Warburg em Oncologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Humanos
8.
Nat Commun ; 12(1): 4974, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404802

RESUMO

Osteoporosis affects millions worldwide and is often caused by osteoclast induced bone loss. Here, we identify the cytoplasmic protein ELMO1 as an important 'signaling node' in osteoclasts. We note that ELMO1 SNPs associate with bone abnormalities in humans, and that ELMO1 deletion in mice reduces bone loss in four in vivo models: osteoprotegerin deficiency, ovariectomy, and two types of inflammatory arthritis. Our transcriptomic analyses coupled with CRISPR/Cas9 genetic deletion identify Elmo1 associated regulators of osteoclast function, including cathepsin G and myeloperoxidase. Further, we define the 'ELMO1 interactome' in osteoclasts via proteomics and reveal proteins required for bone degradation. ELMO1 also contributes to osteoclast sealing zone on bone-like surfaces and distribution of osteoclast-specific proteases. Finally, a 3D structure-based ELMO1 inhibitory peptide reduces bone resorption in wild type osteoclasts. Collectively, we identify ELMO1 as a signaling hub that regulates osteoclast function and bone loss, with relevance to osteoporosis and arthritis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doenças Ósseas Metabólicas/metabolismo , Osteoclastos/metabolismo , Osteoporose/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Artrite/patologia , Reabsorção Óssea/metabolismo , Sistemas CRISPR-Cas , Feminino , Camundongos , Camundongos Knockout , Osteoprotegerina/deficiência , Ovariectomia , Transcriptoma , Microtomografia por Raio-X
9.
Nat Commun ; 12(1): 5023, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408144

RESUMO

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-µbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αß T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-µbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Células-Tronco Hematopoéticas/citologia , Linfopoese , Doenças da Imunodeficiência Primária/terapia , Linfócitos T/citologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD34/genética , Antígenos CD34/imunologia , Proteínas de Ligação ao Cálcio/genética , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/imunologia , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Doenças da Imunodeficiência Primária/fisiopatologia , Linfócitos T/imunologia , Linfócitos T/transplante
10.
Neoplasma ; 68(4): 798-809, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34348465

RESUMO

Osteosarcoma (OS) is a lethal bone malignancy. Circular RNAs (circRNAs) have emerged as important regulators of OS development. CircRNA cyclin dependent kinase 14 (circ_CDK14) was reported to be a potential oncogene in OS. However, the mechanistic pathway by which circ_CDK14 functions in OS is largely unknown. The relative expression of circ_CDK14, microRNA (miR)-520a-3p, and GRB2 Associated Binding Protein 1 (GAB1) was evaluated by quantitative real-time PCR and western blot assays. Flow cytometry was employed to monitor cell cycle distribution and apoptosis. Methyl thiazolyl tetrazolium (MTT) and colony formation assays were performed to assess cell viability and colony formation ability, respectively. Western blot assay was also used to detect the expression of apoptosis-related proteins. Transwell assay was carried out to monitor cell migration and invasion. Additionally, the target association between miR-520a-3p and circ_CDK14 or GAB1 was confirmed by a dual-luciferase reporter assay. Xenograft assay was applied to investigate the role of circ_CDK14 in vivo. Circ_CDK14 and GAB1 expression was upregulated, while miR-520a-3p was downregulated in OS tissues and cells. Circ_CDK14 depletion hindered OS cell proliferation, metastasis, and tumorigenesis while facilitated apoptosis, which were all ameliorated by miR-520a-3p inhibition. Circ_CDK14 could sponge miR-520a-3p. miR-520a-3p targeted GAB1 to repress OS cell proliferation and metastasis. Circ_CDK14 knockdown blocked OS tumor growth in vivo. Circ_CDK14 might positively affect OS development by modulating the miR-520a-3p/GAB1 axis.


Assuntos
MicroRNAs , Osteossarcoma , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Osteossarcoma/genética , RNA Circular
11.
J Genet ; 1002021.
Artigo em Inglês | MEDLINE | ID: mdl-34282730

RESUMO

Choroideraemia (CHM) is a rare X-linked progressive-inherited retinal disease. In this study, we diagnosed and explored the genetic cause in a Chinese pedigree exhibiting nyctalopia and decreased visual acuity in early life. Clinical data and peripheral blood samples were collected from available family members. Sanger sequencing of RPGR and RP2 genes, and subsequently whole-exome sequencing was carried out to investigate the molecular cause. The proband was initially diagnosed as retinitis pigmentosa and experienced night blindness at an early age and decreased visual acuity in teens. The other affected males in this family suffered from the same problem. Direct sequencing failed to reveal the genetic cause and hence a novel hemizygous mutation c.861_862insGCTT was detected by WES in CHM gene resulting in a premature stop codon and a truncated protein. Subsequently, it was confirmed by Sanger sequencing and cosegregation analysis. We describe a novel mutation c.861_862insGCTT in CHM gene in a Chinese pedigree with choroideraemia. Our study emphasizes the utilization of next-generation sequencing in the diagnosis and genetic analysis of retinal diseases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Coroideremia/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Predisposição Genética para Doença , Adolescente , Adulto , Coroideremia/patologia , Códon sem Sentido/genética , Proteínas do Olho/genética , Genes Ligados ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Testes Genéticos , Humanos , Masculino , Mutação/genética , Linhagem , Sequenciamento Completo do Exoma , Adulto Jovem
12.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200497

RESUMO

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFß and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFß/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFß/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFß/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Insuficiência Cardíaca/patologia , Miofibroblastos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Estudos de Casos e Controles , Células Cultivadas , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Miofibroblastos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Ativação Transcricional
13.
Life Sci ; 282: 119820, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34273377

RESUMO

AIMS: It has been demonstrated that miR-145 is expressed in primordial follicles and modulates the initiation of primordial follicle development. We aimed to explore the function of miR-145 in mouse granulosa cells (mGCs). MATERIALS AND METHODS: The proliferation and differentiation of GCs were examined via MTT, EDU assay, QRT-PCR, ELISA and electron microscope analysis. The target of miR-145 was determined by bioinformatics analysis and luciferase reporter assay and the molecular mechanisms were examined via western blot and quantitative Real-Time RT-PCR. KEY FINDINGS: We proved that down-regulation of miR-145 could inhibit GCs proliferation and differentiation. In addition, we provided evidence that Crkl was the target gene of miR-145. The miR-145 antagomir caused an increase in Crkl expression and activation of the JNK/p38 MAPK pathway. Overexpression of Crkl with pEGFP-N1-Crkl vector inhibited GCs differentiation and progesterone synthesis as well as activation of the JNK/p38 MAPK pathway. SIGNIFICANCE: Our study shows that miR-145 targets Crkl and through the JNK/p38 MAPK signaling pathway promotes the GCs proliferation, differentiation, and steroidogenesis. MiR-145 may play an important role in the ovarian physiology and pathology.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Diferenciação Celular , Proliferação de Células , Regulação para Baixo , Células da Granulosa/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Feminino , Camundongos , MicroRNAs/genética
14.
Nat Genet ; 53(8): 1166-1176, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34326544

RESUMO

Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed hybridization chain reaction fluorescence in situ hybridization coupled with flow cytometry (HCR-FlowFISH), a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene and display activating and/or silencing effects. At the cholesterol-level-associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominate causal variants and, importantly, identify their target genes.


Assuntos
Hibridização in Situ Fluorescente/métodos , Sequências Reguladoras de Ácido Nucleico , Proteínas Adaptadoras de Transdução de Sinal/genética , Teorema de Bayes , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Ácidos Graxos Dessaturases/genética , Citometria de Fluxo , Fator de Transcrição GATA1/genética , Humanos , Células K562 , Proteínas com Domínio LIM/genética , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas/genética , Locos de Características Quantitativas , RNA Guia
15.
Arterioscler Thromb Vasc Biol ; 41(9): 2509-2511, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34261329
16.
Cell Death Dis ; 12(7): 664, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215725

RESUMO

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3-/- retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Células Fotorreceptoras de Vertebrados/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Degeneração Retiniana/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagossomos/genética , Autofagossomos/metabolismo , Autofagossomos/patologia , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteínas de Ciclo Celular/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Rodopsina/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
17.
Nat Cell Biol ; 23(7): 758-770, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34226698

RESUMO

The YAP/TAZ transcriptional programme is not only a well-established driver of cancer progression and metastasis but also an important stimulator of tissue regeneration. Here we identified Cerebral cavernous malformations 3 (CCM3) as a regulator of mechanical cue-driven YAP/TAZ signalling, controlling both tumour progression and stem cell differentiation. We demonstrate that CCM3 localizes to focal adhesion sites in cancer-associated fibroblasts, where it regulates mechanotransduction and YAP/TAZ activation. Mechanistically, CCM3 and focal adhesion kinase (FAK) mutually compete for binding to paxillin to fine-tune FAK/Src/paxillin-driven mechanotransduction and YAP/TAZ activation. In mouse models of breast cancer, specific loss of CCM3 in cancer-associated fibroblasts leads to exacerbated tissue remodelling and force transmission to the matrix, resulting in reciprocal YAP/TAZ activation in the neighbouring tumour cells and dissemination of metastasis to distant organs. Similarly, CCM3 regulates the differentiation of mesenchymal stromal/stem cells. In conclusion, CCM3 is a gatekeeper in focal adhesions that controls mechanotransduction and YAP/TAZ signalling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Adesões Focais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mecanotransdução Celular , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Comunicação Celular , Diferenciação Celular , Linhagem Celular Tumoral , Feminino , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/genética , Adesões Focais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Paxilina/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Estresse Mecânico , Fatores de Transcrição/genética , Quinases da Família src/metabolismo
18.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299245

RESUMO

Hepatocellular carcinoma (HCC) records the second-lowest 5-year survival rate despite the avalanche of research into diagnosis and therapy. One of the major obstacles in treatment is chemoresistance to drugs such as 5-fluorouracil (5-FU), making identification and elucidation of chemoresistance regulators highly valuable. As the regulatory landscape grows to encompass non-coding genes such as long non-coding RNAs (lncRNAs), a relatively new class of lncRNA has emerged in the form of pseudogene-derived lncRNAs. Through bioinformatics analyses of the TCGA LIHC dataset, we have systematically identified pseudogenes of prognostic value. Initial experimental validation of selected pseudogene-derived lncRNA (PLEKHA8P1) and its parental gene (PLEKHA8), a well-studied transport protein in Golgi complex recently implicated as an oncogene in both colorectal and liver cancer, indicates that the pseudogene/parental gene pair promotes tumor progression and that their dysregulated expression levels affect 5-FU-induced chemoresistance in human HCC cell line FT3-7. Our study has thus confirmed cancer-related functions of PLEKHA8, and laid the groundwork for identification and validation of oncogenic pseudogene-derived lncRNA that shows potential as a novel therapeutic target in circumventing chemoresistance induced by 5-FU.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Hepatocelular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Bases de Dados Genéticas , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MicroRNAs/genética , Prognóstico , Pseudogenes , RNA Longo não Codificante/genética
19.
Nat Commun ; 12(1): 4502, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301937

RESUMO

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , RNA-Seq/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas do Envelope Viral/genética
20.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34269675

RESUMO

Rabies virus (RABV) infection can initiate the host immune defence response and induce an antiviral state characterized by the expression of interferon (IFN)-stimulated genes (ISGs), among which the family of genes of IFN-induced protein with tetratricopeptide repeats (Ifits) are prominent representatives. Herein, we demonstrated that the mRNA and protein levels of Ifit1, Ifit2 and Ifit3 were highly increased in cultured cells and mouse brains after RABV infection. Recombinant RABV expressing Ifit3, designated rRABV-Ifit3, displayed a lower pathogenicity than the parent RABV in C57BL/6 mice after intramuscular administration, and Ifit3-deficient mice exhibited higher susceptibility to RABV infection and higher mortality during RABV infection. Moreover, compared with their individual expressions, co-expression of Ifit2 and Ifit3 could more effectively inhibit RABV replication in vitro. These results indicate that murine Ifit3 plays an essential role in restricting the replication and reducing the pathogenicity of RABV. Ifit3 acts synergistically with Ifit2 to inhibit RABV replication, providing further insight into the function and complexity of the Ifit family.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Vírus da Raiva/fisiologia , Raiva/virologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Encéfalo/metabolismo , Encéfalo/virologia , Linhagem Celular , Feminino , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Raiva/imunologia , Vírus da Raiva/patogenicidade , Transcriptoma , Carga Viral , Replicação Viral
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