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1.
Cell Mol Life Sci ; 81(1): 283, 2024 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-38963422

RESUMEN

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.


Asunto(s)
AMP Cíclico , Factores de Intercambio de Guanina Nucleótido , Sumoilación , Enzimas Ubiquitina-Conjugadoras , Proteínas de Unión al GTP rap1 , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/química , Humanos , AMP Cíclico/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteínas de Unión al GTP rap1/genética , Células HEK293 , Simulación de Dinámica Molecular , Complejo Shelterina/metabolismo , Transducción de Señal , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/genética , Respuesta al Choque Térmico , Secuencia de Aminoácidos , Unión Proteica
2.
Methods Mol Biol ; 2814: 163-176, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38954205

RESUMEN

Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.


Asunto(s)
Dictyostelium , Proteínas ras , Dictyostelium/metabolismo , Dictyostelium/enzimología , Dictyostelium/genética , Proteínas ras/metabolismo , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal , Unión Proteica
3.
Bioorg Chem ; 147: 107384, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38643568

RESUMEN

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor that is highly susceptible to metastasis, recurrence and resistance, and few therapeutic targets have been identified and proven effective. Herein, we demonstrated for the first time that Rap1b can positively regulate ESCC cell stemness, as well as designed and synthesized a novel class of Pt(IV) complexes that can effectively inhibit Raplb. In vitro biological studies showed that complex-1 exhibited stronger cytotoxicity than cisplatin and oxaliplatin against a variety of ESCC cells, and effectively reversed cisplatin-induced resistance of TE6 cells by increasing cellular accumulation of platinum and inhibiting cancer cell stemness. Significantly, complex-1 also exhibited strong ability to reversal cisplatin-induced cancer cell resistance and inhibit tumor growth in TE6/cDDP xenograft mice models, with a tumor growth inhibition rate of 73.3 % at 13 mg/kg and did not show significant systemic toxicity. Overall, Rap1b is a promising target to be developed as an effective treatment for ESCC. Complex-1, as the first Pt(IV) complex that can strongly inhibit Rap1b, is also worthy of further in-depth study.


Asunto(s)
Antineoplásicos , Proliferación Celular , Cisplatino , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Cisplatino/farmacología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Ratones , Proliferación Celular/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Relación Estructura-Actividad , Estructura Molecular , Relación Dosis-Respuesta a Droga , Ligandos , Ratones Desnudos , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/antagonistas & inhibidores , Ratones Endogámicos BALB C , Compuestos Organoplatinos/farmacología , Compuestos Organoplatinos/química , Compuestos Organoplatinos/síntesis química , Línea Celular Tumoral , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neoplasias Experimentales/metabolismo , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química
4.
J Biol Chem ; 300(5): 107257, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38574891

RESUMEN

The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo signaling pathway activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivation. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.


Asunto(s)
Vía de Señalización Hippo , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Animales , Humanos , Ratones , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Paclitaxel/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Fosforilación
5.
J Gastroenterol ; 59(2): 119-137, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37925679

RESUMEN

BACKGROUND: Three-dimensional (3D) chromatin architecture frequently altered in cancer. However, its changes during the pathogenesis of hepatocellular carcinoma (HCC) remained elusive. METHODS: Hi-C and RNA-seq were applied to study the 3D chromatin landscapes and gene expression of HCC and ANHT. Hi-C Pro was used to generate genome-wide raw interaction matrices, which were normalized via iterative correction (ICE). Moreover, the chromosomes were divided into different compartments according to the first principal component (E1). Furthermore, topologically associated domains (TADs) were visualized via WashU Epigenome Browser. Furthermore, differential expression analysis of ANHT and HCC was performed using the DESeq2 R package. Additionally, dysregulated genes associated with 3D genome architecture altered were confirmed using TCGA, qRT-PCR, immunohistochemistry (IHC), etc. RESULTS: First, the intrachromosomal interactions of chr1, chr2, chr5, and chr11 were significantly different, and the interchromosomal interactions of chr4-chr10, chr13-chr21, chr15-chr22, and chr16-chr19 are remarkably different between ANHT and HCC, which resulted in the up-regulation of TP53I3 and ZNF738 and the down-regulation of APOC3 and APOA5 in HCC. Second, 49 compartment regions on 18 chromosomes have significantly switched (A-B or B-A) during HCC tumorigenesis, contributing to up-regulation of RAP2A. Finally, a tumor-specific TAD boundary located on chr5: 6271000-6478000 and enhancer hijacking were identified in HCC tissues, potentially associated with the elevated expression of MED10, whose expression were associated with poor prognosis of HCC patients. CONCLUSION: This study demonstrates the crucial role of chromosomal structure variation in HCC oncogenesis and potential novel biomarkers of HCC, laying a foundation for cancer precision medicine development.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patología , Cromatina/genética , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/patología , Cromosomas/metabolismo , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Complejo Mediador/genética , Complejo Mediador/metabolismo
6.
Clin Genet ; 105(2): 196-201, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-37850357

RESUMEN

Syndromic constitutive thrombocytopenia encompasses a heterogeneous group of disorders characterised by quantitative and qualitative defects of platelets while featuring other malformations. Recently, heterozygous, de novo variants in RAP1B were reported in three cases of syndromic thrombocytopenia. Here, we report two additional, unrelated individuals identified retrospectively in our data repository with heterozygous variants in RAP1B: NM_001010942.2(RAP1B):c.35G>A, p.(Gly12Glu) (de novo) and NM_001010942.2(RAP1B):c.178G>A, p.(Gly60Arg). Both individuals had thrombocytopenia, as well as congenital malformations, and neurological, behavioural, and dysmorphic features, in line with previous reports. Our data supports the causal role of monoallelic RAP1B variants that disrupt RAP1B GTPase activity in syndromic congenital thrombocytopenia.


Asunto(s)
Plaquetas , Trombocitopenia , Humanos , Estudios Retrospectivos , Plaquetas/metabolismo , Trombocitopenia/genética , Proteínas de Unión al GTP rap
7.
Aging (Albany NY) ; 15(21): 12275-12295, 2023 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-37934565

RESUMEN

Glioma is a common intracranial tumor and is generally associated with poor prognosis. Recently, numerous studies illustrated the importance of 5-methylcytosine (m5C) RNA modification to tumorigenesis. However, the prognostic value and immune correlation of m5C in glioma remain unclear. We obtained RNA expression and clinical information from The Cancer Genome Atlas (TCGA) and The Chinese Glioma Genome Atlas (CGGA) datasets to analyze. Nonnegative matrix factorization (NMF) was used to classify patients into two subgroups and compare these patients in survival and clinicopathological characteristics. CIBERSORT and single-sample gene-set algorithm (ssGSEA) methods were used to investigate the relationship between m5C and the immune environment. The Weighted correlation network analysis (WGCNA) and univariate Cox proportional hazard model (CoxPH) were used to construct a m5C-related signature. Most of m5C RNA methylation regulators presented differential expression and prognostic values. There were obvious relationships between immune infiltration cells and m5C regulators, especially NSUN7. In the m5C-related module from WGCNA, we found SEPT3, CHI3L1, PLBD1, PHYHIPL, SAMD8, RAP1B, B3GNT5, RER1, PTPN7, SLC39A1, and MXI1 were prognostic factors for glioma, and they were used to construct the signature. The great significance of m5C-related signature in predicting the survival of patients with glioma was confirmed in the validation sets and CGGA cohort.


Asunto(s)
Neoplasias Encefálicas , Glioma , Humanos , Metilación , Glioma/genética , Pronóstico , Neoplasias Encefálicas/genética , ARN , Microambiente Tumoral/genética , Proteínas de Unión al GTP rap
8.
Int J Mol Sci ; 24(18)2023 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-37762144

RESUMEN

Osteopetrosis is a rare inherited disease caused by osteoclast failure, resulting in increasing bone density in humans. Patients with osteopetrosis possess several dental and cranial complications. Since carbonic anhydrase II (CA-II) deficiency is a major cause of osteopetrosis, CA-II activators might be an attractive potential treatment option for osteopetrosis patients. We conducted comprehensive label-free quantitative proteomics analysis on Fluconazole-treated Dental Pulp Mesenchymal Stem/Stromal Cells from CA-II-Deficient Osteopetrosis Patients. We identified 251 distinct differentially expressed proteins between healthy subjects, as well as untreated and azole-treated derived cells from osteopetrosis patients. Twenty-six (26) of these proteins were closely associated with osteogenesis and osteopetrosis disease. Among them are ATP1A2, CPOX, Ap2 alpha, RAP1B and some members of the RAB protein family. Others include AnnexinA1, 5, PYGL, OSTF1 and PGAM4, all interacting with OSTM1 in the catalytic reactions of HCO3 and the Cl- channel via CAII regulation. In addition, the pro-inflammatory/osteoclast regulatory proteins RACK1, MTSE, STING1, S100A13, ECE1 and TRIM10 are involved. We have identified proteins involved in osteogenic and immune metabolic pathways, including ERK 1/2, phosphatase and ATPase, which opens the door for some CA activators to be used as an alternative drug therapy for osteopetrosis patients. These findings propose that fluconazole might be a potential treatment agent for CAII- deficient OP patients. Altogether, our findings provide a basis for further work to elucidate the clinical utility of azole, a CA activator, as a therapeutic for OP.


Asunto(s)
Células Madre Mesenquimatosas , Osteopetrosis , Humanos , Fluconazol/farmacología , Fluconazol/uso terapéutico , Osteogénesis , Pulpa Dental , Osteopetrosis/tratamiento farmacológico , Azoles , Redes y Vías Metabólicas , Proteínas de Unión al GTP rap
9.
Front Endocrinol (Lausanne) ; 14: 1162786, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37621654

RESUMEN

Introduction: Endometriosis, a benign inflammatory disease whereby endometrial-like tissue grows outside the uterus, is a risk factor for endometriosis-associated ovarian cancers. In particular, ovarian endometriomas, cystic lesions of deeply invasive endometriosis, are considered the precursor lesion for ovarian clear-cell carcinoma (OCCC). Methods: To explore this transcriptomic landscape, OCCC from women with pathology-proven concurrent endometriosis (n = 4) were compared to benign endometriomas (n = 4) by bulk RNA and small-RNA sequencing. Results: Analysis of protein-coding genes identified 2449 upregulated and 3131 downregulated protein-coding genes (DESeq2, P< 0.05, log2 fold-change > |1|) in OCCC with concurrent endometriosis compared to endometriomas. Gene set enrichment analysis showed upregulation of pathways involved in cell cycle regulation and DNA replication and downregulation of pathways involved in cytokine receptor signaling and matrisome. Comparison of pathway activation scores between the clinical samples and publicly-available datasets for OCCC cell lines revealed significant molecular similarities between OCCC with concurrent endometriosis and OVTOKO, OVISE, RMG1, OVMANA, TOV21G, IGROV1, and JHOC5 cell lines. Analysis of miRNAs revealed 64 upregulated and 61 downregulated mature miRNA molecules (DESeq2, P< 0.05, log2 fold-change > |1|). MiR-10a-5p represented over 21% of the miRNA molecules in OCCC with endometriosis and was significantly upregulated (NGS: log2fold change = 4.37, P = 2.43e-18; QPCR: 8.1-fold change, P< 0.05). Correlation between miR-10a expression level in OCCC cell lines and IC50 (50% inhibitory concentration) of carboplatin in vitro revealed a positive correlation (R2 = 0.93). MiR-10a overexpression in vitro resulted in a significant decrease in proliferation (n = 6; P< 0.05) compared to transfection with a non-targeting control miRNA. Similarly, the cell-cycle analysis revealed a significant shift in cells from S and G2 to G1 (n = 6; P< 0.0001). Bioinformatic analysis predicted that miR-10a-5p target genes that were downregulated in OCCC with endometriosis were involved in receptor signaling pathways, proliferation, and cell cycle progression. MiR-10a overexpression in vitro was correlated with decreased expression of predicted miR-10a target genes critical for proliferation, cell-cycle regulation, and cell survival including [SERPINE1 (3-fold downregulated; P< 0.05), CDK6 (2.4-fold downregulated; P< 0.05), and RAP2A (2-3-fold downregulated; P< 0.05)]. Discussion: These studies in OCCC suggest that miR-10a-5p is an impactful, potentially oncogenic molecule, which warrants further studies.


Asunto(s)
Adenocarcinoma de Células Claras , Endometriosis , MicroARNs , Humanos , Femenino , Endometriosis/complicaciones , Endometriosis/genética , Transcriptoma , MicroARNs/genética , Perfilación de la Expresión Génica , Adenocarcinoma de Células Claras/complicaciones , Adenocarcinoma de Células Claras/genética , Proteínas de Unión al GTP rap
10.
DNA Cell Biol ; 42(10): 617-637, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37610843

RESUMEN

Recent studies have shown that several members of the G-protein-coupled receptors (GPCR) superfamily play crucial roles in the maintenance of ion-water homeostasis of the sperm and Sertoli cells, development of the germ cells, formation of the blood barrier, and maturation of sperm. The GPCR, guanyl-nucleotide exchange factor, membrane traffic protein, and small GTPase genes were analyzed by microarray and bioinformatics (3513 sperm and Sertoli cell genes). In the microarray analyses of three human cases with different nonobstructive azoospermia sperm, the expression of GOLGA8IP, OR2AT4, PHKA1, A2M, OR56A1, SEMA3G, LRRC17, APP, ARHGAP33, RABGEF1, NPY2R, GHRHR, LTB4R2, GRIK5, OR6K6, NAPG, OR6C65, VPS35, FPR3, and ARL4A was upregulated, while expression of MARS, SIRPG, OGFR, GPR150, LRRK1, and NGEF was downregulated. There was an increase in GBP3, GBP3, TNF, TGFB3, and CLTC expression in the Sertoli cells of three human cases with NOA, whereas expression of PAQR4, RRAGD, RAC2, SERPINB8, IRPB1, MRGPRF, RASA2, SIRPG, RGS2, RAP2A, RAB2B, ARL17, SERINC4, XIAP, DENND4C, ANKRA2, CSTA, STX18, and SNAP23 were downregulated. A combined analysis of Enrich Shiny Gene Ontology (GO), STRING, and Cytoscape was used to predict proteins' molecular interactions and then to recognize master pathways. Functional enrichment analysis showed that the biological process (BP), regulation of protein metabolic process, regulation of small GTPase-mediated signal transduction were significantly expressed in up-/downregulated differentially expressed genes (DEGs) in sperm. In molecular function (MF) experiments of DEGs that were up-/downregulated, it was found that GPCR activity, guanyl ribonucleotide binding, GTPase activity and nucleoside-triphosphatase activity were overexpressed. An analysis of GO enrichment findings of Sertoli cells showed BP and MF to be common DEGs. When these gene mutations have been validated, they can be used to create new GPCR antagonists or agonists that are receptor-selective.


Asunto(s)
Azoospermia , Proteínas de Unión al GTP Monoméricas , Humanos , Masculino , Testículo/metabolismo , Azoospermia/genética , Azoospermia/metabolismo , Semen/metabolismo , Expresión Génica , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas Activadoras de ras GTPasa/genética , Ancirinas/genética , Ancirinas/metabolismo , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo
11.
J Cancer Res Clin Oncol ; 149(13): 11647-11659, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37405477

RESUMEN

BACKGROUND: Cuproptosis, a form of copper-dependent programmed cell death recently presented by Tsvetkov et al., have been identified as a potential therapeutic target for refractory cancers and ferroptosis, a well-known form describing iron-dependent cell death. However, whether the crossing of cuproptosis-related genes and ferroptosis-related genes can introduce some new idea, thus being used as a novel clinical and therapeutic predictor in esophageal squamous cell carcinoma (ESCC) remains unknown. METHODS: We collected ESCC patient data from the Gene Expression Omnibus and the Cancer Genome Atlas databases and used Gene Set Variation Analysis to score each sample based on cuproptosis and ferroptosis. We then performed weighted gene co-expression network analysis to identify cuproptosis and ferroptosis-related genes (CFRGs) and construct a ferroptosis and cuproptosis-related risk prognostic model, which we validated using a test group. We also investigated the relationship between the risk score and other molecular features, such as signaling pathways, immune infiltration, and mutation status. RESULTS: Four CFRGs (MIDN, C15orf65, COMTD1 and RAP2B) were identified to construct our risk prognostic model. Patients were classified into low- and high-risk groups based on our risk prognostic model and the low-risk group showed significantly higher survival possibilities (P < 0.001). We used the "GO", "cibersort" and "ESTIMATE" methods to the above-mentioned genes to estimate the relationship among the risk score, correlated pathways, immune infiltration, and tumor purity. CONCLUSION: We constructed a prognostic model using four CFRGs and demonstrated its potential clinical and therapeutic guidance value for ESCC patients.


Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Ferroptosis , Humanos , Carcinoma de Células Escamosas de Esófago/genética , Pronóstico , Ferroptosis/genética , Neoplasias Esofágicas/genética , Apoptosis , Proteínas de Unión al GTP rap
12.
Ocul Surf ; 29: 68-76, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37094778

RESUMEN

PURPOSE: To identify specific dry eye disease (DED) tear biomarker(s) using tear proteomic analysis, clinical parameters, and their correlations before and after DED treatment. METHODS: A prospective, double-blinded, national multicenter clinical study was performed using data from 80 DED patients. The patients were treated with 0.1% cyclosporine (CsA, n = 28), 0.05% CsA (n = 26), or 3% diquafosol (DQS, n = 26) eye drops, and tear proteome changes and clinical outcomes (tear break-up time [TBUT], corneal erosion [Cor-Er], conjunctival erosion [Conj-Er], and symptom assessment in dry eye [SANDE] scores) were observed at 4, 8, and 12 weeks. For all clinical parameters, correlation analysis was performed between the three drug conditions and the differentially expressed proteins (DEPs) from the proteomic analysis. RESULTS: AFM, ALCAM, CFB, H1-4, PON1, RAP1B, and RBP4 were identified in all treatment groups and were downregulated after treatment. All clinical parameters significantly improved at 12 weeks than at baseline (p-value <0.0001); however, their values were not significantly different among groups, except for Cor-Er (p-value = 0.007). Compared with the DQS group, Cor-Er score significantly improved after treatment with 0.1% and 0.05% CsA. The seven DEPs identified in all groups were not consistently correlated with the clinical parameters (p-value >0.05). CONCLUSIONS: Despite differences in drug concentration and action mechanisms, the improvement levels of TBUT, Cor-Er, and SANDE scores were clinically adequate. However, useful tear protein biomarkers, clinically acceptable biomarker combinations correlating with clinical parameters, and clinically acceptable levels of specificity and sensitivity were not identified.


Asunto(s)
Úlcera de la Córnea , Síndromes de Ojo Seco , Humanos , Proteómica , Estudios Prospectivos , Ciclosporina/uso terapéutico , Síndromes de Ojo Seco/diagnóstico , Síndromes de Ojo Seco/tratamiento farmacológico , Síndromes de Ojo Seco/metabolismo , Biomarcadores , Arildialquilfosfatasa/metabolismo , Arildialquilfosfatasa/uso terapéutico , Proteínas Plasmáticas de Unión al Retinol , Proteínas de Unión al GTP rap/metabolismo
13.
Acta Biochim Biophys Sin (Shanghai) ; 55(3): 382-393, 2023 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-36951484

RESUMEN

Acute myeloid leukemia (AML) is a myeloid malignancy with generally high mortality. Although recent advances in AML research have revealed that circRNAs play significant roles in AML progression, our understanding of the leukemogenic mechanism of circRNAs remains very limited. In this study, increased expression of hsa_circ_0013880 was observed in bone marrow mononuclear cells (BMNCs) of AML patients. Overexpression of hsa_circ_0013880 promotes AML cell proliferation and migration and reduces cell apoptosis. Mechanistically, hsa_circ_0013880 could elevate the expression of USP32, a deubiquitinating enzyme that is highly expressed in the BMNCs of AML patients. Given the deubiquitination function of USP32, we further hypothesize that USP32 may mediate the malignant behaviors of AML cells by regulating the stability of Ras-related protein (Rap1b). At the molecular level, we find that silencing of USP32 increases ubiquitinated Rap1b. Overexpression of Rap1b restores the effects of USP32 knockdown, which further verifies our hypothesis. In addition, we propose another hypothesis that a potential regulatory network among hsa_circ_0013880, miR-148a-3p/miR-20a-5p and USP32 might exist in the development of AML, according to bioinformatics website predictions and our preliminary experimental verification. Overall, our findings will enrich the understanding of the hsa_circ_0013880/USP32/Rap1b axis in AML development, which may contribute to the development of novel therapeutic strategies for AML.


Asunto(s)
Leucemia Mieloide Aguda , MicroARNs , Humanos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Unión al GTP rap/metabolismo , ARN Circular/genética , ARN Circular/metabolismo
14.
Urol Int ; 107(6): 632-645, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36958293

RESUMEN

INTRODUCTION: Circular RNAs (circRNAs) are related to the pathogenesis and progression of bladder cancer (BC). This research aimed to investigate the role and mechanism of hsa_circ_0008035 (circ_0008035) in BC progression. METHODS: Circ_0008035, microRNA (miR)-1,184, and Ras-related protein 2B (RAP2B) levels were examined in BC via quantitative real-time polymerase chain reaction and Western blotting. Cell Counting Kit-8, colony formation, 5-ethynyl-2'-deoxyuridine staining, flow cytometry, caspase-3 assay kit, transwell, and tube formation assays were conducted to estimate the effects of circ_0008035 on the malignant phenotypes of BC tumors. The interaction between RNAs and genes was evaluated via a dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft model of BC in nude mice was established to estimate the effect of circ_0008035 in BC in vivo. RESULTS: Circ_0008035 and RAP2B levels were upregulated, while miR-1184 abundance was downregulated in BC tissues and cells. Circ_0008035 knockdown constrained cell proliferation, migration, invasion and angiogenesis but promoted apoptosis in vitro. And circ_0008035 silencing curbed xenograft tumor growth in vivo. Circ_0008035 acted as a miRNA sponge for miR-1184. Circ_0008035 increased RAP2B expression by sponging miR-1184. MiR-1184 downregulation relieved the effects of circ_0008035 knockdown on BC progression. And RAP2B knockdown partly reversed the effects of miR-1184 overexpression on BC progression. CONCLUSION: Circ_0008035-mediated BC progression via regulating the miR-1184/RAP2B axis, providing a potential target for BC treatment.


Asunto(s)
MicroARNs , Neoplasias de la Vejiga Urinaria , Humanos , Animales , Ratones , Ratones Desnudos , Neoplasias de la Vejiga Urinaria/genética , Vejiga Urinaria , Apoptosis , Vendajes , Proliferación Celular , MicroARNs/genética , Proteínas de Unión al GTP rap
15.
Angiogenesis ; 26(2): 265-278, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36403190

RESUMEN

Overcoming vascular immunosuppression: lack of endothelial cell (EC) responsiveness to inflammatory stimuli in the proangiogenic environment of tumors, is essential for successful cancer immunotherapy. The mechanisms through which Vascular Endothelial Growth Factor A(VEGF-A) modulates tumor EC response to exclude T-cells are not well understood. Here, we demonstrate that EC-specific deletion of small GTPase Rap1B, previously implicated in normal angiogenesis, restricts tumor growth in endothelial-specific Rap1B-knockout (Rap1BiΔEC) mice. EC-specific Rap1B deletion inhibits angiogenesis, but also leads to an altered tumor microenvironment with increased recruitment of leukocytes and increased activity of tumor CD8+ T-cells. Depletion of CD8+ T-cells restored tumor growth in Rap1BiΔEC mice. Mechanistically, global transcriptome and functional analyses indicated upregulation of signaling by a tumor cytokine, TNF-α, and increased NF-κB transcription in Rap1B-deficient ECs. Rap1B-deficiency led to elevated proinflammatory chemokine and Cell Adhesion Molecules (CAMs) expression in TNF-α stimulated ECs. Importantly, CAM expression was elevated in tumor ECs from Rap1BiΔEC mice. Significantly, Rap1B deletion prevented VEGF-A-induced immunosuppressive downregulation of CAM expression, demonstrating that Rap1B is essential for VEGF-A-suppressive signaling. Thus, our studies identify a novel endothelial-endogenous mechanism underlying VEGF-A-dependent desensitization of EC to proinflammatory stimuli. Significantly, they identify EC Rap1B as a potential novel vascular target in cancer immunotherapy.


Asunto(s)
Linfocitos T CD8-positivos , Células Endoteliales , Neoplasias , Proteínas de Unión al GTP rap , Animales , Ratones , Linfocitos T CD8-positivos/inmunología , Terapia de Inmunosupresión , Neoplasias/irrigación sanguínea , Neoplasias/genética , Neoplasias/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunología , Células Endoteliales/inmunología , Células Endoteliales/fisiología , FN-kappa B/genética , FN-kappa B/inmunología , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/inmunología
16.
Clin Breast Cancer ; 23(1): 71-83, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36289041

RESUMEN

BACKGROUND: The therapeutic effect of adriamycin (ADM) has been limited by chemoresistance in breast cancer (BC). Circular RNAs are involved in resistance regulation by mediating the miRNA/mRNA axis. Circ_0001667 enhanced ADM resistance via the miR-4458/NCOA3 axis in BC. This study was to investigate the other miRNA/mRNA network for circ_0001667. METHODS: The level detection of circ_0001667, microRNA-193a-5p (miR-193a-5p) or Ras-Related Protein 2a (Rap2A) was conducted by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Half inhibitory concentration (IC50) of ADM was detected through cell counting kit-8 (CCK-8) assay. The proliferation analysis was performed by colony formation assay and EdU assay. Flow cytometry was used for assessing apoptosis. Transwell assay was applied for examining cell migration and invasion. The protein detection was carried out by western blot. In vivo assay was performed using xenograft tumor model. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were implemented to validate the target interaction. RESULTS: Circ_0001667 was highly expressed in ADM-resistant BC tissues and cells. Downregulation of circ_0001667 reduced ADM resistance and inhibited proliferation, migration, invasion in ADM-resistant BC cells. Tumor growth was repressed by circ_0001667 knockdown in ADM-resistant xenograft model. Circ_0001667 has induced the sponge effect on miR-193a-5p. The circ_0001667 function was partly achieved by targeting miR-193a-5p. Rap2A expression was positively regulated by circ_0001667 through sponging miR-193a-5p. The miR-193a-5p upregulation restrained chemoresistance and BC progression by the downregulation of Rap2A. CONCLUSION: All results unraveled that circ_0001667 contributed to ADM resistance and tumor development in BC via the miR-193a-5p-mediated Rap2A expression change, providing a novel regulatory mechanism for circ_0001667.


Asunto(s)
Neoplasias de la Mama , MicroARNs , ARN Circular , Proteínas de Unión al GTP rap , Femenino , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proliferación Celular , Regulación hacia Abajo , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , MicroARNs/genética , Regulación hacia Arriba , ARN Circular/genética
17.
J Dent Res ; 102(3): 302-312, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36366779

RESUMEN

Abnormal stress loading has been considered a major contributor to the initiation of temporomandibular joint osteoarthritis (TMJOA), but studies to date have not identified a functional molecule that transforms physical stress into biological or biochemical signaling in chondrocytes in response to excessive mechanical stress. Ras-related protein Rap-2a (RAP2A) is reportedly a molecular switch that relays extracellular matrix rigidity signals via the Hippo/Yes-associated protein (YAP) pathway. In the present study, RAP2A diminished with cartilage degradation in unilateral anterior crossbite-induced TMJOA mice, as well as severe cartilage matrix degeneration and TMJOA formation in Cre-loxP-mediated conditional RAP2A knockout mice. RAP2A in chondrocytes regulated the Hippo/YAP pathway directly in response to matrix stiffness, and RAP2A/Hippo/YAP was critical for a chondrocyte phenotype switch and matrix synthesis function. Loss of RAP2A impaired cartilage homeostasis and altered chondrocyte phenotype via Hippo/YAP/SRY-box transcription factor 9 signaling. It may be possible to generate therapeutic strategies using RAP2A or YAP to attenuate the TMJOA pathological process at an early stage. This is the first study to reveal the molecular function of RAP2A in TMJOA progression as a mechanotransduction molecule in condylar chondrocytes.


Asunto(s)
Cartílago Articular , Osteoartritis , Ratones , Animales , Mecanotransducción Celular , Cartílago Articular/patología , Transducción de Señal , Articulación Temporomandibular/metabolismo , Condrocitos/metabolismo , Osteoartritis/patología , Proteínas de Unión al GTP rap/metabolismo
18.
Int J Mol Sci ; 23(23)2022 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-36499729

RESUMEN

Cutaneous squamous cell carcinoma (CSCC) is an epidermal skin cancer that evolves from normal epidermis along several pre-malignant stages. Previously we found specific miRNAs alterations in each step along these stages. miR-199a-3p expression decreases at the transition to later stages. A crucial step for epithelial carcinoma cells to acquire invasive capacity is the disruption of cell-cell contacts and the gain of mesenchymal motile phenotype, a process known as epithelial-to-mesenchymal transition (EMT). This study aims to study the role of decreased expression of miR-199a-3p in keratinocytes' EMT towards carcinogenesis. First, we measured miR-199a-3p in different stages of epidermal carcinogenesis. Then, we applied Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) assay to search for possible biochemical targets of miR-199a-3p and verified that Ras-associated protein B2 (RAP2B) is a bona-fide target of miR-199a-3p. Next, we analyzed RAP2B expression, in CSCC biopsies. Last, we evaluated possible mechanisms leading to decreased miR-199a-3p expression. miR-199a-3p induces a mesenchymal to epithelial transition (MET) in CSSC cells. Many of the under-expressed genes in CSCC overexpressing miR-199a-3p, are possible targets of miR-199a-3p and play roles in EMT. RAP2B is a biochemical target of miR-199a-3p. Overexpression of miR-199a-3p in CSCC results in decreased phosphorylated focal adhesion kinase (FAK). In addition, inhibiting FAK phosphorylation inhibits EMT marker genes' expression. In addition, we proved that DNA methylation is part of the mechanism by which miR-199a-3p expression is inhibited. However, it is not by the methylation of miR-199a putative promoter. These findings suggest that miR-199a-3p inhibits the EMT process by targeting RAP2B. Inhibitors of RAP2B or FAK may be effective therapeutic agents for CSCC.


Asunto(s)
Carcinoma de Células Escamosas , MicroARNs , Neoplasias Cutáneas , Humanos , Carcinoma de Células Escamosas/patología , Regulación Neoplásica de la Expresión Génica , Proteínas ras/metabolismo , Línea Celular Tumoral , Neoplasias Cutáneas/patología , MicroARNs/genética , MicroARNs/metabolismo , Transición Epitelial-Mesenquimal/genética , Proliferación Celular , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo
19.
Front Immunol ; 13: 1061544, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36505495

RESUMEN

Introduction: Neutrophils are critical for host immune defense; yet, aberrant neutrophil tissue infiltration triggers tissue damage. Neutrophils are heterogeneous functionally, and adopt 'normal' or 'pathogenic' effector function responses. Understanding neutrophil heterogeneity could provide specificity in targeting inflammation. We previously identified a signaling pathway that suppresses neutrophilmediated inflammation via integrin-mediated Rap1b signaling pathway. Methods: Here, we used Rap1-deficient neutrophils and proteomics to identify pathways that specifically control pathogenic neutrophil effector function. Results: We show neutrophil acidity is normally prevented by Rap1b during normal immune response with loss of Rap1b resulting in increased neutrophil acidity via enhanced Ldha activity and abnormal neutrophil behavior. Acidity drives the formation of abnormal invasive-like protrusions in neutrophils, causing a shift to transcellular migration through endothelial cells. Acidity increases neutrophil extracellular matrix degradation activity and increases vascular leakage in vivo. Pathogenic inflammatory condition of ischemia/reperfusion injury is associated with increased neutrophil transcellular migration and vascular leakage. Reducing acidity with lactate dehydrogenase inhibition in vivo limits tissue infiltration of pathogenic neutrophils but less so of normal neutrophils, and reduces vascular leakage. Discussion: Acidic milieu renders neutrophils more dependent on Ldha activity such that their effector functions are more readily inhibited by small molecule inhibitor of Ldha activity, which offers a therapeutic window for antilactate dehydrogenase treatment in specific targeting of pathogenic neutrophils in vivo.


Asunto(s)
Células Endoteliales , Neutrófilos , Humanos , Movimiento Celular , Infiltración Neutrófila , Inflamación , L-Lactato Deshidrogenasa , Proteínas de Unión al GTP rap
20.
Acta Biochim Pol ; 69(4): 719-724, 2022 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-36444911

RESUMEN

Retinoblastoma generally affects children and causes permanent vision failure or even death. MicroRNAs (miRs) have recently gained much attention during recent years. The miR-708 acts as a tumor suppressor in several human cancers, but the former has not been functionally characterized in human retinoblastoma. The present study was designed to investigate the role of miR-708 in human retinoblastoma. The results showed that miR-708 is significantly (P<0.05) downregulated in retinoblastoma cell lines. MiR-708 overexpression significantly (P<0.05) inhibited retinoblastoma cell growth and proliferation by inducing apoptosis. Furthermore, retinoblastoma cells overexpressing miR-708 exhibited a markedly lower migratory rate and invasiveness compared to negative control cells. The bioinformatics and dual luciferase assay revealed a RAS oncogene family protein, RAP2B, which acts as the regulatory target and functional mediator of the molecular role of miR-708 in retinoblastoma. Together, the present study revealed the tumor suppressor role of miR-708 and pointed to the therapeutic implications of miR-708/RAP2B in the treatment of retinoblastoma.


Asunto(s)
MicroARNs , Neoplasias de la Retina , Retinoblastoma , Proteínas de Unión al GTP rap , Niño , Humanos , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Genes ras , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Neoplasias de la Retina/genética , Neoplasias de la Retina/patología , Retinoblastoma/genética , Retinoblastoma/patología
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