RESUMEN
BACKGROUND: Extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs) have shown therapeutic effects on liver fibrosis. This study aimed to evaluate the effects of extracellular vesicles from placenta-derived MSCs (Pd-MSCs-EVs) on liver fibrosis at 3D/2D levels and explore the potential mechanisms. METHODS: The multicellular liver organoids, consisting of hepatocytes, hepatic stellate cells (HSCs), Kupffer cells, and liver sinusoidal endothelial cells, were observed for growth status, morphological changes, and metabolism. Human transformation growth factor- beta 1 (TGF-ß1) was used to induce fibrosis at optimal concentration. The anti-fibrosis effects of Pd-MSCs-EVs were evaluated in liver organoids and HSCs models. Anti-fibrotic content of Pd-MSCs-EVs was identified by multiple experimental validations. RESULTS: TGF-ß1 induced fibrosis in liver organoids, while Pd-MSCs-EVs significantly alleviated fibrotic phenotypes. Following serial verifications, miR-378c was identified as a potential key anti-fibrosis content. In contrast, miR-378c depletion decreased the anti-fibrotic effects of Pd-MSCs-EVs. Additionally, Pd-MSCs-EVs administration repressed TGF-ß1-mediated HSCs activation at 2D or 3D levels. Mechanistically, exosomal miR-378c inactivated HSCs by inhibiting epithelial-mesenchymal transition (EMT) through stabilizing E-cadherin via targeting its E3 ubiquitin ligase S-Phase Kinase Associated Protein 2 (SKP2). CONCLUSION: Pd-MSCs-EVs ameliorated TGF-ß1-induced fibrosis by deactivating HSCs in a miR-378c/SKP2-dependent manner, which may be an efficient therapeutic candidate for liver fibrosis.
RESUMEN
Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence-based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase-1 [ALAS-1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS-1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, ß-estradiol, hydroxychloroquine sulfate, alpha-methyldopa, D (-) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi-well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.
Asunto(s)
Fenitoína , Ácido Valproico , Técnicas de Cultivo de Célula , Hemo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Norgestrel/metabolismo , Fenobarbital/metabolismo , Fenobarbital/farmacología , Fenitoína/metabolismo , Porfobilinógeno Sintasa/deficiencia , Porfirias Hepáticas , Ácido Valproico/metabolismoRESUMEN
Generating microliver tissues to recapitulate hepatic function is of increasing importance in tissue engineering and drug screening. But the limited availability of primary hepatocytes and the marked loss of phenotype hinders their application. Human induced hepatocytes (hiHeps) generated by direct reprogramming can address the shortage of primary hepatocytes to make personalized drug prediction possible. Here, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Transducing human fetal and adult fibroblasts with low vector dosage generated human induced hepatocyte-like cells (hiHeps) displaying characteristics of mature hepatocytes and capable of drug metabolism. To mimic the physiologic liver microenvironment and improve hepatocyte function, we prepared 3D scaffold-free microliver spheroids using hiHeps and human liver nonparenchymal cells through self-assembly without exogenous scaffolds. We then introduced the microliver spheroids into a two-organ microfluidic system to examine interactions between hepatocytes and tumor cells. The hiHeps-derived spheroids metabolized the prodrug capecitabine into the active metabolite 5-fluorouracil and induced toxicity in downstream tumor spheroids. Our results demonstrate that hiHeps can be used to make microliver spheroids and combined with a microfluidic system for drug evaluation. Our work could make it possible to use patient-specific hepatocyte-like cells to predict drug efficacy and side effects in various organs from the same patient.
Asunto(s)
Hepatocitos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Adulto , Reprogramación Celular , Fibroblastos , Humanos , Esferoides Celulares , Ingeniería de Tejidos , Factores de TranscripciónRESUMEN
BACKGROUND: Organoids play an important role in basic research, drug screening, and regenerative medicine. Here, we aimed to develop a novel kind of three-dimensional (3D) organoids generated from urine-derived stem cells (USCs) and to explore whether kidney-specific extracellular matrix (kECM) could enable such organoids for renal function in vitro. METHODS: USCs were isolated from human urine samples and cultured with kECM extraction to generate 3D organoids in vitro. Eight densities from 1000 to 8000 cells per organoids were prepared, and both ATP assay and Live/Dead staining were used to determine the optimal USC density in forming organoids and kECM additive concentration. The morphology and histology of as-made organoids were evaluated by hematoxylin and eosin (H.E.) staining, immunofluorescence staining and whole mount staining. Additionally, RT-qPCR was implemented to detect renal-related gene expression. Drug toxicity test was conducted to evaluate the potential application for drug screening. The renal organoids generated from whole adult kidney cells were used as a positive control in multiple assessments. RESULTS: The optimized cell density to generate ideal USC-derived organoids (USC-organoids) was 5000 cells/well, which was set as applying density in the following experiments. Besides, the optimal concentration of kECM was revealed to be 10%. On this condition, Live/Dead staining showed that USC-organoids were well self-organized without significant cell death. Moreover, H.E. staining showed that compact and viable organoids were generated without obvious necrosis inside organoids, which were very close to renal organoids morphologically. Furthermore, specific proximal tubule marker Aquaporin-1 (AQP1), kidney endocrine product erythropoietin (EPO), kidney glomerular markers Podocin and Synaptopodin were detected positively in USC-organoids with kECM. Nephrotoxicity testing showed that aspirin, penicillin G, and cisplatin could exert drug-induced toxicity on USC-organoids with kECM. CONCLUSIONS: USC-organoids could be developed from USCs via an optimal procedure. Combining culture with kECM, USC-organoid properties including morphology, histology, and specific gene expression were identified to be similar with real renal organoids. Additionally, USC-organoids posed kECM in vitro showed the potential to be a drug screening tool which might take the place of renal organoids to some extent in the future.
Asunto(s)
Organoides , Sistema Urinario , Adulto , Cisplatino , Humanos , Riñón , Células MadreRESUMEN
The kidney function of patients with chronic kidney disease (CKD) is impaired irreversibly. Organ transplantation is the only treatment to restore kidney function in CKD patients. The assessment of new potential therapeutic procedures relies heavily on experimental animal models, but it is limited by its human predictive capacity. In addition, the frequently used two-dimensional in vitro human renal cell models cannot replicate all the features of the in vivo situation. In this study, we developed a three-dimensional (3D) in vitro human renal organoid model from whole kidney cells as a promising drug screening tool. At present, the renal tissue generated from human pluripotent stem cells (hPSCs) exhibits intrinsic tumorigenicity properties. Here we first developed a 3D renal organoid culture system that originated from adult differentiated cells without gene modification. Renal organoids composed of multiple cell types were created under optimal experimental conditions and evaluated for morphology, viability and erythropoietin production. As a novel screening tool for renal toxicity, 3D organoids were exposed to three widely used drugs: aspirin, penicillin G and cisplatin. The study results showed this 3D renal organoid model can be used as a drug screening tool, a new in vitro 3D human kidney model, and provide hope for potential regenerative therapies for CKD.
Asunto(s)
Riñón/citología , Organoides/citología , Células Madre Pluripotentes/citología , Aspirina/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Cultivadas , Cisplatino/farmacología , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Organoides/metabolismo , Organoides/fisiología , Penicilina G/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Current drug development techniques are expensive and inefficient, partially due to the use of preclinical models that do not accurately recapitulate in vivo drug efficacy and cytotoxicity. To address this challenge, we report on an integrated, in vitro multi-organoid system that enables parallel assessment of drug efficiency and toxicity on multiple 3D tissue organoids. Built in a low-cost, adhesive film-based microfluidic device, these miniaturized structures require less than 200 µL fluid volume and are amenable to both matrix-based 3D cell culture and spheroid aggregate integration, each supported with an in situ photocrosslinkable hyaluronic acid hydrogel. Here, we demonstrate this technology first with a three-organoid device consisting of liver, cardiac, and lung constructs. We show that these multiple tissue types can be kept in common circulation with high viability for 21 days and validate the platform by investigating liver metabolism of the prodrug capecitabine into 5-fluorouracil (5-FU) and observing downstream toxicity in lung and cardiac organoids. Then we expand the integrated system to accommodate six humanized constructs, including liver, cardiac, lung, endothelium, brain, and testes organoids. Following a 14-day incubation in common media, we demonstrate multi-tissue interactions by metabolizing the alkylating prodrug ifosfamide in the liver organoid to produce chloroacetaldehyde and induce downstream neurotoxicity. Our results establish an expandable, multi-organoid body-on-a-chip system that can be fabricated easily and used for the accurate characterization of drug interactions in vitro. STATEMENT OF SIGNIFICANCE: The use of 3-dimensional (3D) in vitro models in drug development has advanced over the past decade. However, with several exceptions, the majority of research studies using 3D in vitro models, such as organoids, employ single tissue types, in isolated environments with no "communication" between different tissues. This is a significant limiting factor because in the human body there is significant signaling between different cells, tissues, and organs. Here we employ a low-cost, adhesive film-based microfluidic device approach, paired with a versatile extracellular matrix-derived hyaluronic acid hydrogel to support integrated systems of 3 and 6 3D organoid and cell constructs. Moreover, we demonstrate an integrated response to drugs, in which downstream toxicity is dependent on the presence of liver organoids.
Asunto(s)
Capecitabina/metabolismo , Ifosfamida/metabolismo , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Organoides/metabolismo , Profármacos/metabolismo , Capecitabina/toxicidad , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácido Hialurónico/química , Hidrogeles/química , Ifosfamida/toxicidad , Organoides/efectos de los fármacos , Profármacos/toxicidadRESUMEN
To investigate the role of miR-122 in the development and regression of non-alcoholic fatty liver disease (NAFLD) in vitro, we used multicellular 3D human liver organoids developed in our laboratory. These organoids consist of primary human hepatocytes, Kupffer cells, quiescent stellate cells and liver sinusoidal endothelial cells. They remain viable and functional for 4 weeks expressing typical markers of liver function such as synthesis of albumin, urea, and alpha-1 p450 drug metabolism. Before mixing, hepatic cells were transduced with lentivirus to inhibit miR122 expression (ABM, CA). Immediately after the organoids were fully formed (day 4) or after 1 or 2 weeks of additional incubation (days 11 or 18), the organoids were analyzed using fluorescent live/dead staining and ATP production; total RNA was extracted for qPCR gene expression profiling. Our results show that miR-122 inhibition in liver organoids leads to inflammation, necrosis, steatosis and fibrosis. This was associated with increase in inflammatory cytokines (IL6, TNF), chemokines (CCL2, CCL3) and increase in a subset of Matrix Metaloproteinases (MMP8, MMP9). An altered expression of key genes in lipid metabolism (i.e LPL, LDLR) and insulin signaling (i.e GLUT4, IRS1) was also identified. CONCLUSION: Our results highlight the role of miR-122 inhibition in liver inflammation, steatofibrosis and dysregulation of insulin signaling. Patients with NAFLD are known to have altered levels of miR-122, therefore we suggest that miR-122 mimics could play a useful role in reversing liver steatofibrosis and insulin resistance seen in patients with NAFLD.
Asunto(s)
Inflamación/metabolismo , Insulina/metabolismo , Hígado/citología , Hígado/metabolismo , MicroARNs/metabolismo , Necrosis/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Organoides/citología , Organoides/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Hepatocitos/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Interleucina-6/metabolismo , Macrófagos del Hígado/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Transducción de SeñalRESUMEN
SOX7 is a transcription factor and acts as a tumor suppressor, but its target genes in cancers are poorly explored. We revealed SOX7-mediated gene expression profile in breast cancer cells using microarray chips and discovered multiple altered signaling pathways. When combinatorially analyzing the microarray data with a gene array dataset from 759 breast cancer patients, we identified four genes as potential targets of SOX7 and validated them by quantitative PCR and chromatin immunoprecipitation assays. Among these four genes, we determined that SOX7-activated SPRY1 and SLIT2, and SOX7-repressed TRIB3 and MTHFD2 could all differentially contribute to SOX7-mediated tumor suppression. Overall, we identified multiple cancer-related pathways mediated by SOX7 and for the first time revealed SOX7-regulated target genes in a cancer-relevant context.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Factores de Transcripción SOXF/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Genes Reporteros , Humanos , Unión Proteica , Reproducibilidad de los Resultados , TranscriptomaRESUMEN
Fatty acid synthase (FASN) is upregulated in breast cancer and correlates with poor prognosis. FASN contributes to mammary oncogenesis and serves as a bona fide target in cancer therapies. MicroRNAs inhibit gene expression through blocking mRNA translation or promoting mRNA degradation by targeting their 3'-UTRs. We identified four microRNAs in two microRNA clusters miR-15a-16-1 and miR-497-195 that share a common seed sequence to target the 3'-UTR of the FASN mRNA. In reporter assays, both of these microRNA clusters inhibited the expression of a reporter construct containing the FASN 3'-UTR. However, only ectopic miR-15a-16-1, but not miR-497-195, markedly reduced the levels of endogenous FASN in breast cancer cells. Both miR-15a and miR-16-1 contributes to inhibiting FASN expression and breast cancer cell proliferation. Consistently, a sponge construct consisting of eight repeats of the FASN 3'-UTR region targeted by these microRNAs could markedly increase endogenous FASN levels in mammary cells. When FASN expression was restored by ectopic expression in breast cancer cells, retarded cell proliferation caused by miR-15a-16-1 was partially rescued. In conclusion, we demonstrated that FASN expression is primarily downregulated by miR-15a and miR-16-1 in mammary cells and FASN is one of the major targets of these two tumor suppressive microRNAs.
Asunto(s)
Neoplasias de la Mama/genética , Acido Graso Sintasa Tipo I/genética , MicroARNs/genética , Regiones no Traducidas 3' , Sitios de Unión , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proliferación Celular , Regulación hacia Abajo , Acido Graso Sintasa Tipo I/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , MicroARNs/metabolismo , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.
Asunto(s)
Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción YY1/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Modelos Biológicos , Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Dominios Proteicos , Relación Estructura-Actividad , Factor de Transcripción YY1/químicaRESUMEN
Both epigenetic silencing and genetic deletion of tumor suppressors contribute to the development and progression of breast cancer. SOX7 is a transcription factor important to development, and its down-regulation has been reported in tumor tissues and cell lines of prostate, colon, and lung cancers. However, the regulation of SOX7 expression and its functional role in breast cancer have not been reported. The current study demonstrates that SOX7 mRNA and protein expression are down-regulated in breast cancer tissues and cell lines compared with adjacent normal tissues and nontumorigenic cells, respectively. The SOX7 promoter is hypermethylated in breast cancer cell lines compared with nontumorigenic cells, and the inhibition of DNA methylation increases SOX7 mRNA levels. With shRNA-mediated SOX7 silencing, nontumorigenic immortal breast cells display increased proliferation, migration, and invasion and form structures that resemble that of breast cancer cells in a three-dimensional culture system. Conversely, ectopic SOX7 expression inhibits proliferation, migration, and invasion of breast cancer cells in vitro and tumor growth in vivo. Importantly, we discovered that SOX7 transcript levels positively correlated with clinical outcome of 674 breast cancer patients. Overall, our data suggest that SOX7 acts as a tumor suppressor in breast cancer. SOX7 expression is likely regulated by multiple mechanisms and potentially serves as a prognostic marker for breast cancer patients.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción SOXF/genética , Proteínas Supresoras de Tumor/genética , Animales , Línea Celular Tumoral , Metilación de ADN/genética , Regulación hacia Abajo/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Desnudos , Regiones Promotoras Genéticas , Factores de Transcripción SOXF/metabolismo , Resultado del Tratamiento , Proteínas Supresoras de Tumor/metabolismoRESUMEN
RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.
Asunto(s)
Neoplasias de la Mama/genética , ADN/genética , Vectores Genéticos/genética , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Transfección/métodos , Animales , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Células HEK293 , Humanos , Lentivirus/genética , Ratones , Ratones Desnudos , Reacción en Cadena de la Polimerasa/métodos , ARN Interferente Pequeño/genética , Factor de Transcripción YY1/genéticaRESUMEN
Yin Yang 1 (YY1) is highly expressed in various types of cancers and regulates tumorigenesis through multiple pathways. In the present study, we evaluated YY1 expression levels in breast cancer cell lines, a breast cancer TMA, and two gene arrays. We observed that, compared with normal samples, YY1 is generally overexpressed in breast cancer cells and tissues. In functional studies, depletion of YY1 inhibited the clonogenicity, migration, invasion, and tumor formation of breast cancer cells, but did not affect the clonogenicity of nontumorigenic cells. Conversely, ectopically expressed YY1 enhanced the migration and invasion of nontumorigenic MCF-10A breast cells. In both a monolayer culture condition and a three-dimensional Matrigel system, silenced YY1 expression changed the architecture of breast cancer MCF-7 cells to that resembling MCF-10A cells, whereas ectopically expressed YY1 in MCF-10A cells had the opposite effect. Furthermore, we detected an inverse correlation between YY1 and p27 expression in both breast cancer cells and xenograft tumors with manipulated YY1 expression. Counteracting the changes in p27 expression attenuated the effects of YY1 alterations on these cells. In addition, YY1 promoted p27 ubiquitination and physically interacted with p27. In conclusion, our data suggest that YY1 is an oncogene and identify p27 as a new target of YY1.
Asunto(s)
Neoplasias de la Mama/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factor de Transcripción YY1/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ciclo Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Forma de la Célula/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Trasplante de Neoplasias , Células Madre Neoplásicas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Antígeno Nuclear de Célula en Proliferación/genética , Procesamiento Proteico-Postraduccional/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Trasplante Heterólogo , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiología , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismoRESUMEN
Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.
Asunto(s)
Regiones no Traducidas 5' , ARN Helicasas DEAD-box/metabolismo , G-Cuádruplex , Regiones Promotoras Genéticas , Recombinasas/metabolismo , Factor de Transcripción YY1/genética , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Cationes Monovalentes/química , Línea Celular , Dicroismo Circular , ADN/química , Huella de ADN , Femenino , Secuencia Rica en GC , Expresión Génica , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN/química , Factor de Transcripción YY1/metabolismoRESUMEN
BACKGROUND: In prostate cancer (PCa), the common treatment involving androgen ablation alleviates the disease temporarily, but results in the recurrence of highly aggressive and androgen-independent metastatic cancer. Therefore, more effective therapeutic approaches are needed. It is known that aberrant epigenetics contributes to prostate malignancy. Unlike genetic changes, these epigenetic alterations are reversible, which makes them attractive targets in PCa therapy to impede cancer progression. As a histone methyltransferase, Ezh2 plays an essential role in epigenetic regulation. Since Ezh2 is overexpressed and acts as an oncogene in PCa, it has been proposed as a bona fide target of PCa therapy. MicroRNAs (miRNAs) regulate gene expression through modulating protein translation. Recently, the contribution of miRNAs in cancer development is increasingly appreciated. In this report, we present our study showing that microRNA-101 (miR-101) inhibits Ezh2 expression and differentially regulates prostate cancer cells. In addition, the expression of miR-101 alters upon androgen treatment and HIF-1alpha/HIF-1beta induction. RESULT: In our reporter assays, both miR-101 and miR-26a inhibit the expression of a reporter construct containing the 3'-UTR of Ezh2. When ectopically expressed in PC-3, DU145 and LNCaP cells, miR-101 inhibits endogenous Ezh2 expression in all three cell lines, while miR-26a only decreases Ezh2 in DU145. Ectopic miR-101 reduces the invasion ability of PC-3 cells, while restored Ezh2 expression rescues the invasiveness of PC-3 cells. Similarly, miR-101 also inhibits cell invasion and migration of DU145 and LNCaP cells, respectively. Interestingly, ectopic miR-101 exhibits differential effects on the proliferation of PC-3, DU-145 and LNCaP cells and also causes morphological changes of LNCaP cells. In addition, the expression of miR-101 is regulated by androgen receptor and HIF-1alpha/HIF-1beta. While HIF-1alpha/HIF-1beta induced by deferoxamine mesylate (DFO) decreases miR-101 levels, the overall effects of R-1881 on miR-101 expression are stimulatory. CONCLUSIONS: This study indicates that miR-101 targets Ezh2 and decreases the invasiveness of PCa cells, suggesting that miR-101 introduction is a potential therapeutic strategy to combat PCa. MiR-101 differentially regulates prostate cell proliferation. Meanwhile, the expression of miR-101 is also modulated at different physiological conditions, such as androgen stimulation and HIF-1alpha/HIF-1beta induction.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Proteínas de Unión al ADN/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MicroARNs/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Factores de Transcripción/biosíntesis , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Invasividad Neoplásica/genética , Complejo Represivo Polycomb 2 , Neoplasias de la Próstata/genética , Receptores Androgénicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , TransfecciónRESUMEN
As a multifunctional protein, Yin Yang 1 (YY1) has been demonstrated to regulate both gene expression and protein posttranslational modifications. However, gaps still exist in our knowledge of how YY1 can be modified and what the consequences of its modifications are. Here we report that YY1 protein can be sumoylated both in vivo and in vitro. We have identified lysine 288 as the major sumoylation site of YY1. We also discovered that PIASy, a SUMO E3 ligase, is a novel YY1-interacting protein and can stimulate the sumoylation of YY1 both in vitro and in vivo. Importantly, the effects of PIASy mutants on in vivo YY1 sumoylation correlate with the YY1-PIASy interaction but do not depend on the RING finger domain of PIASy. This regulation is unique to YY1 sumoylation because PIASy-mediated p53 sumoylation still relies on the integrity of PIASy, which is also true of all of the previously identified substrates of PIASy. In addition, PIASy colocalizes with YY1 in the nucleus, stabilizes YY1 in vivo, and differentially regulates YY1 transcriptional activity on different target promoters. This study demonstrates that YY1 is a target of SUMOs and reveals a novel feature of a SUMO E3 ligase in the PIAS family that selectively stimulates protein sumoylation independent of the RING finger domain.
Asunto(s)
Proteínas Inhibidoras de STAT Activados/metabolismo , Proteína SUMO-1/metabolismo , Factor de Transcripción YY1/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Regulación de la Expresión Génica , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Inhibidoras de STAT Activados/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SUMO-1/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Factor de Transcripción YY1/genéticaRESUMEN
NK cell receptors (NKRs) modulate T lymphocyte responses by modifying the Ag activation threshold. However, what governs their expression on T cells remains unclear. In this study we show that different NKRs are imprinted on CD8 T cells in the gut mucosa and periphery during the same Ag challenge. After a viral, bacterial, and tumor challenge, most CD8 peritoneal exudate lymphocytes expressed NKG2A but not 2B4. In contrast, most CD8 intraepithelial lymphocytes exhibited 2B4 but not NKG2A. Our data suggest that tissue-specific factors may determine the pattern of NKR expression. In the gut, CD70 licensing appears to promote 2B4 induction on mucosal CD8 T cells. Conversely, retinoic acid produced by the intestinal dendritic cells may suppress NKG2A expression. Thus, tissue-specific factors regulate NKR expression and may confer T cells with differing effector functions in a tissue and site-specific manner.