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SUMMARY: Esophageal cancer is one of the most aggressive gastrointestinal cancers. Invasion and metastasis are the main causes of poor prognosis of esophageal cancer. SPRY2 has been reported to exert promoting effects in human cancers, which controls signal pathways including PI3K/AKT and MAPKs. However, the expression of SPRY2 in esophageal squamous cell carcinoma (ESCC) and its underlying mechanism remain unclear. In the present study, we aimed to investigate the detailed role of SPRY2 in the regulation of cell proliferation, invasion and ERK/AKT signaling pathway in ESCC. It was identified that the expression level of SPRY2 in ESCC was remarkably decreased compared with normal tissues, and it was related to clinicopathologic features and prognosis ESCC patients. The upregulation of SPRY2 expression notably inhibited the proliferation, migration and invasion of Eca-109 cells. In addition, the activity of ERK /AKT signaling was also suppressed by the SPRY2 upregulation in Eca-109 cells. Our study suggests that overexpression of SPRY2 suppress cancer cell proliferation and invasion of by through suppression of the ERK/AKT signaling pathways in ESCC. Therefore, SPRY2 may be a promising prognostic marker and therapeutic target for ESCC.
El cáncer de esófago es uno de los cánceres gastrointestinales más agresivos. La invasión y la metástasis son las principales causas de mal pronóstico del cáncer de esófago. Se ha informado que SPRY2 ejerce efectos promotores en los cánceres humanos, que controla las vías de señales, incluidas PI3K/AKT y MAPK. Sin embargo, la expresión de SPRY2 en el carcinoma de células escamosas de esófago (ESCC) y su mecanismo subyacente aún no están claros. En el presente estudio, nuestro objetivo fue investigar el papel detallado de SPRY2 en la regulación de la proliferación celular, la invasión y la vía de señalización ERK/AKT en ESCC. Se identificó que el nivel de expresión de SPRY2 en ESCC estaba notablemente disminuido en comparación con los tejidos normales, y estaba relacionado con las características clínico-patológicas y el pronóstico de los pacientes con ESCC. La regulación positiva de la expresión de SPRY2 inhibió notablemente la proliferación, migración e invasión de células Eca-109. Además, la actividad de la señalización de ERK/AKT también fue suprimida por la regulación positiva de SPRY2 en las células Eca-109. Nuestro estudio sugiere que la sobreexpresión de SPRY2 suprime la proliferación y la invasión de células cancerosas mediante la supresión de las vías de señalización ERK/AKT en ESCC. Por lo tanto, SPRY2 puede ser un marcador de pronóstico prometedor y un objetivo terapéutico para la ESCC.
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Humanos , Neoplasias Esofágicas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Carcinoma de Células Escamosas de Esófago/metabolismo , Proteínas de la Membrana/metabolismo , Inmunohistoquímica , Biomarcadores de Tumor , Western Blotting , Quinasas MAP Reguladas por Señal Extracelular , Proliferación Celular , Proteínas Proto-Oncogénicas c-aktRESUMEN
CONTEXT: Wen-Shen-Tong-Luo-Zhi-Tong (WSTLZT) Decoction is a Chinese prescription with antiosteoporosis effects, especially in patients with abnormal lipid metabolism. OBJECTIVE: To explore the effect and mechanism of WSTLZT on osteoporosis (OP) through adipocyte-derived exosomes. MATERIALS AND METHODS: Adipocyte-derived exosomes with or without WSTLZT treated were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blotting (WB). Co-culture experiments for bone marrow mesenchymal stem cells (BMSCs) and exosomes were performed to examine the uptake and effect of exosome in osteogenesis and adipogenic differentiation of BMSC. MicroRNA profiles, luciferase and IP were used for exploring specific mechanisms of exosome on BMSC. In vivo, 80 Balb/c mice were randomly divided into four groups: Sham, Ovx, Exo (30 µg exosomes), Exo-WSTLZT (30 µg WSTLZT-exosomes), tail vein injection every week. After 12 weeks, the bone microstructure and marrow fat distribution were analysed by micro-CT. RESULTS: ALP, Alizarin red and Oil red staining showed that WSTLZT-induced exosomes from adipocyte can regulate osteoblastic and adipogenic differentiation of BMSC. MicroRNA profiles observed that WSTLZT treatment resulted in 87 differentially expressed miRNAs (p < 0.05). MiR-122-5p with the greatest difference was screened by q-PCR (p < 0.01). The target relationship between miR-122-5p and SPRY2 was tested by luciferase and IP. MiR-122-5p negatively regulated SPRY2 and elevated the activity of MAPK signalling pathway, thereby regulating the osteoblastic and adipogenic differentiation of BMSC. In vivo, exosomes can not only improve bone microarchitecture but also significantly reduce accumulation of bone marrow adipose. CONCLUSIONS: WSTLZT can exert anti-OP effect through SPRY2 via the MAKP signalling by miR-122-5p carried by adipocyte-derived exosomes.
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Exosomas , MicroARNs , Ratones , Animales , Exosomas/genética , Exosomas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Diferenciación Celular , Adipogénesis , Osteogénesis , AdipocitosRESUMEN
Crocin has been reported to have antitumor activity in several tumors including breast cancer. Nevertheless, the mechanism of action of crocin on breast cancer remains unclear. The cytotoxicity of crocin was evaluated by CCK-8 assay. Cell proliferation was assessed using EdU incorporation assay and western blot analysis. Breast cancer-related genes were extracted from GEPIA. miR-122-5p targets were predicted using Targetscan, starbase, and miRDB softwares. Luciferase reporter assay was employed to confirm whether miR-122-5p targeted sprouty2 (SPRY2) and forkhead box P2 (FOXP2). Results showed that crocin exhibited cytotoxicity and suppressed the proliferation in breast cancer cells. miR-122-5p was upregulated in breast cancer tissues and cells. Crocin suppressed miR-122-5p to block the proliferation of breast cancer cells. Seven targets of miR-122-5p were identified in breast cancer. SPRY2 and FOXP2 were selected for further experiments due to their involvement in breast cancer. miR-122-5p targeted SPRY2 and FOXP2 to inhibit their expression. miR-122-5p knockdown restrained breast cancer cell proliferation by targeting SPRY2 and FOXP2. Additionally, crocin increased SPRY2 and FOXP2 expression by inhibiting miR-122-5p expression. Together, our results suggested that crocin inhibited proliferation of breast cancer cells through decreasing miR-122-5p expression and the subsequent increase of SPRY2 and FOXP2 expression.
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Neoplasias de la Mama , MicroARNs , Humanos , Femenino , MicroARNs/genética , MicroARNs/metabolismo , Carcinógenos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismoRESUMEN
OBJECTIVE: Neuroblastoma is one of the most common extracranial solid tumors in children. The forkhead transcription factor FOXO3a has been implicated in the progression of a variety of human diseases. Here, we aim to identify the effects of FOXO3a on the malignancy of neuroblastoma. METHODS: Bioinformatics analysis was employed to identify differentially expressed genes related to neuroblastoma and the downstream regulator of FOXO3a. FOXO3a expression was examined in SH-SY5Y neuroblastoma cells. Interactions between FOXO3a and microRNA-21 (miR-21) were then identified using bioinformatics analysis and dual-luciferase reporter assay. After ectopic expression and depletion experiments in SH-SY5Y cells, cell malignant phenotypes were assessed by cell counting kit-8 and Transwell assays. FOXO3a-overexpressing neuroblastoma cells were xenografted into nude mice to validate the role of FOXO3a in tumor growth. RESULTS: Downregulated expression of FOXO3a was observed in neuroblastoma cells, with a negative correlation between FOXO3a and miR-21 expression. FOXO3a bound to the promoter region of miR-21 to downregulate its expression, resulting in inhibition of SH-SY5Y cell malignant phenotypes. Additionally, miR-21 targeted SPRY2 by binding to the 3'UTR of the mRNA encoding SPRY2, activating the extracellular signal-regulated kinase (ERK) pathway. FOXO3a disrupted the binding of miR-21 to SPRY2 and inactivated ERK to suppress the malignant phenotypes of SH-SY5Y cells as well as tumor growth in vivo. CONCLUSIONS: In conclusion, FOXO3a may inhibit the progression of neuroblastoma by suppressing the miR-21 expression and facilitating SPRY2-dependent ERK pathway inactivation.
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Proteína Forkhead Box O3/genética , MicroARNs , Neuroblastoma , Animales , Línea Celular Tumoral , Proliferación Celular , Niño , Quinasas MAP Reguladas por Señal Extracelular/genética , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Neuroblastoma/metabolismoRESUMEN
BACKGROUND AND AIMS: Human vascular smooth muscle cells (HA-VSMCs) are an important cell type involved in atherosclerosis. Low density lipoprotein (LDL) is a lipoprotein particle that carries cholesterol into peripheral tissue cells, and oxidized modified LDL (ox-LDL) is a well-known inducer of the atherosclerosis-related phenotype switch in VSMCs, leading to the occurrence of atherosclerosis. Accumulating studies have revealed that long non-coding RNAs (lncRNAs) mediate the effect of ox-LDL on the atherosclerosis-related biological activities of HA-VSMCs, including proliferation, migration, and apoptosis. However, the mechanism of small nucleolar RNA host gene 12 (SNHG12) in ox-LDL-induced phenotype switch of VSMCs remains unclear. Thus, this research dug in whether SNHG12 mediated the influence of ox-LDL on HA-VSMCs and the potential mechanism. METHODS: Fundamental experiments and functional assays were performed to measure the function of SNHG12 on HA-VSMCs. Then, mechanism assays and rescue assays were performed to study the regulatory mechanism of SNHG12 in HA-VSMCs. RESULTS: SNHG12 reversed the influence of ox-LDL treatment in enhancing cell proliferative and migratory abilities and weakening apoptotic ability in HA-VSMCs. SNHG12 was a competitive endogenous RNA (ceRNA) competing with sprouty RTK signaling antagonist 2 (SPRY2) to bind to miR-1301-3p, thus up-regulating SPRY2 expression in ox-LDL-treated HA-VSMCs. Besides, SNHG12 recruited serine and arginine rich splicing factor 1 (SRSF1) to stabilize negative regulator of ubiquitin like proteins 1 (NUB1) expression. CONCLUSIONS: This study illustrated that SNHG12 inhibited cell proliferation, migration and facilitated cell apoptosis in ox-LDL-induced HA-VSMCs by up-regulating SPRY2 and NUB1.
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MicroARNs , Músculo Liso Vascular , ARN Largo no Codificante , Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Movimiento Celular , Proliferación Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas LDL , Proteínas de la Membrana , Miocitos del Músculo Liso , Regulación hacia ArribaRESUMEN
Numerous studies have reported the regulatory effects of miR-21-5p and reversine in human breast cancer (HBC). However, the mechanism of reversine and miR-21-5p has not been fully investigated in HBC. The aim of the current study was to assess the mechanism of action of reversine, with or without miR-21-5p, in HBC progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot results confirmed the upregulation of miR-21-5p and downregulation of sprouty RTK signaling antagonist 2 (SPRY2) in HBC. Bioinformatics analysis and luciferase assay identified the correlation between miR-21-5p and SPRY2. Cell function experiment results indicated a decrease in migration, proliferation, and invasion of HBC cells treated with miR-21-5p inhibitor and reversine; however, an increase in apoptosis was observed in these cells. Apoptotic ability was more enhanced and migration, proliferation, and invasion were more impaired in HBC cells treated with both miR-21-5p inhibitor and reversine than in those treated individually with either inhibitors. SPRY2, downstream of miR-21-5p, participated in HBC progression with reversine. Overall, our study proved that combining the miR-21-5p inhibitor with reversine produced a synergistic effect by regulating SPRY2, thereby limiting HBC progression. This knowledge might offer insights into the clinical therapy of HBC.
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Antagomirs/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , MicroARNs/genética , Morfolinas/administración & dosificación , Purinas/administración & dosificación , Animales , Antagomirs/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Células MCF-7 , Ratones , MicroARNs/antagonistas & inhibidores , Morfolinas/farmacología , Estadificación de Neoplasias , Purinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Conventional wisdom is that Sprouty2 (SPRY2), a suppressor of Receptor Tyrosine Kinase (RTK) signaling, functions as a tumor suppressor and is downregulated in many solid tumors. We reported, for the first time, that increased expression of SPRY2 augments cancer phenotype and Epithelial-Mesenchymal-Transition (EMT) in colorectal cancer (CRC). In this report, we assessed epigenetic DNA modifications that regulate SPRY2 expression in CRC. A total of 4 loci within SPRY2 were evaluated for 5mC using Combined Bisulfite Restriction Analysis (COBRA). Previously sequenced 5hmC nano-hmC seal data within SPRY2 promoter and gene body were evaluated in CRC. Combined bioinformatics analyses of SPRY2 CRC transcripts by RNA-seq/microarray and 450K methyl-array data archived in The Cancer Genome Atlas (TCGA) and GEO database were performed. SPRY2 protein in CRC tumors and cells was measured by Western blotting. Increased SPRY2 mRNA was observed across several CRC datasets and increased protein expression was observed among CRC patient samples. For the first time, SPRY2 hypomethylation was identified in adenocarcinomas in the promoter and gene body. We also revealed, for the first time, increases of 5hmC deposition in the promoter region of SPRY2 in CRC. SPRY2 promoter hypomethylation and increased 5hmC may play an influential role in upregulating SPRY2 in CRC.
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Neoplasias Colorrectales/genética , Epigénesis Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Regulación hacia Arriba/genética , 5-Metilcitosina/metabolismo , Adenoma/genética , Sitios de Unión/genética , Factor de Unión a CCCTC/metabolismo , Línea Celular Tumoral , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Activating transcription factor 2(ATF2), a transcription factor belonging to the AP-1 family, plays an important role in inflammation. However, its biological functions and underlying molecular mechanisms in rheumatoid arthritis (RA) remain unclear. Western blot and immunohistochemistry were used to identify the expression of ATF2 and Sprouty2(SPRY2) in RA synovial tissues. SW982 cells were stimulated by TNF-α to establish an in vitro RA fibroblast-like synoviocyte (RA-FLS) model. Transwell and monolayer wound-healing were used to detect cell migration and invasion. RNA interference (si-ATF2) and adenovirus vector (Ad-SPRY2) methods were employed to manipulate ATF2 or SPRY2 expression in SW982 cells. The protein expression and phosphorylation levels in SW982 cells were evaluated by western blot. ATF2 expression and phosphorylation were upregulated in the RA synovial tissues. In RA-FLS model, ATF2 expression and phosphorylation were increased in a time-dependent manner. ATF2 knockdown inhibited the migration and invasion of RA-FLS model, reducing the inflammatory factors, which was consistent with the influence on cell behaviors caused by SPRY2 overexpression. Moreover, SPRY2 overexpression inhibited the TNF-α-induced phosphorylation of ERK and ATF2 in SW982 cells. The high expression and phosphorylation of ATF2 promoted migration and invasion of RA-FLSs. SPRY2 might inhibited the inflammatory responses of RA-FLSs via suppressing ERK-ATF2 pathway.
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Factor de Transcripción Activador 2/biosíntesis , Artritis Reumatoide/metabolismo , Movimiento Celular/fisiología , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas de la Membrana/biosíntesis , Sinoviocitos/metabolismo , Factor de Transcripción Activador 2/antagonistas & inhibidores , Factor de Transcripción Activador 2/genética , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Células Cultivadas , Regulación hacia Abajo/fisiología , Fibroblastos/patología , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Fosforilación/fisiología , Sinoviocitos/patologíaRESUMEN
BACKGROUND: It has been documented that microRNAs (miRs) assume a pivotal role in the development of ischemic stroke (IS). However, it remains poorly identified about the role of miR-122 in IS. Herein, this study was intended to explore the mechanism of E2F1-orchestrated miR-122 in IS. PATIENTS AND METHODS: E2F1, miR-122, and SPRY2 expression in serum from patients with IS and oxygen-glucose deprivation (OGD)-treated N2a cells was detected by RT-qPCR. After gain- and loss-of-function approaches in OGD-induced N2a cells, GAFP staining, flow cytometry, and Western blot analysis were adopted to assess neuronal viability, cell cycle and apoptosis, and expression of apoptosis- and autophagy-related proteins, respectively. Meanwhile, mice with IS were induced, in which E2F1, miR-122, and SPRY2 were overexpressed, followed by evaluation of neurological deficit and cerebral infarction area. The MAPK pathway activity in tissues of mice and cells was determined. RESULTS: miR-122 was down-regulated, and E2F1 and SPRY2 were up-regulated in IS patients and OGD-induced N2a cells. E2F1 inhibited miR-122 transcription, while miR-122 targeted SPRY2. Overexpression (OE) of miR-122 or down-regulation of E2F1 or SPRY2 increased viability, but decreased apoptosis, cell cycle arrest, and autophagy in OGD-induced N2a cells. In IS mice, the neurological deficit score and cerebral infarction area were elevated, which was aggravated by up-regulating E2F1 or SPRY2 but attenuated by overexpressing miR-122. E2F1/miR-122/SPRY2 axis mediated the MAPK pathway in vivo and in vitro. CONCLUSION: Collectively, E2F1 reduced miR-122 transcription to up-regulate SPRY2, which inactivated MAPK pathway and promoted neurological deficit in IS.
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PURPOSE: The mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. Because the increased activity and expression of epidermal growth factor receptor (EGFR) kinase have been reported in A549 cancer cells overexpressing CUG2 (A549-CUG2) compared with control cells (A549-Vec), the Sprouty2 (Spry2) protein has gained attention as the downstream molecule of EGFR signaling. Therefore, we aim to identify the role of Spry2 in CUG2-overexpressing lung cancer cells. MATERIALS AND METHODS: Spry2 expression levels were examined in A549-CUG2 and A549-Vec cells by Western blotting and qRT-PCR. Cell migration, invasion, and sphere formation were examined after Spry2 suppression and overexpression. EGFR-Stat1 and Akt-ERK protein phosphorylation levels were detected via immunoblotting. NEK2 kinase and ß-catenin reporter assay were performed for downstream of Spry2 signaling. RESULTS: Although A549-CUG2 cells showed lower levels of the Spry2 protein than A549-Vec cells, no difference in levels of Spry2 transcript was observed between both cells via qRT-PCR. Furthermore, MG132 treatment enhanced the protein levels and ubiquitination of Spry2, suggesting that Spry2 protein expression can be regulated via the ubiquitin-proteasome pathway. The enforced expression of c-Cbl, known as the binding partner of Spry2, decreased the Spry2 protein levels, whereas its knockdown oppositely increased them. Epithelial-mesenchymal transition (EMT) and sphere formation were increased in A549-Vec cells during Spry2 siRNA treatment, confirming the role of Spry2 in CUG2-induced oncogenesis. Furthermore, EMT and sphere formation were determined by the Spry2 protein levels through the regulation of EGFR-Stat1 and ß-catenin-NEK2-Yap1 signaling pathways. CONCLUSION: CUG2 reduces Spry2 protein levels, the negative signaling molecule of cell proliferation, via c-Cbl, possibly activating the EGFR and ß-catenin signaling pathways and, in turn, contributing to the induction of cancer stem cell-like phenotypes.
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As a reversible scar repair reaction, liver fibrosis can be blocked or even reversed by proper intervention during its formation. Our work suggests that acid-sensitive ion channel 1a (ASIC1a) participates in liver fibrosis and presents a novel mechanism involving m6 A modification and miR-350/SPRY2. We demonstrated that the expression of ASIC1a was significantly increased in liver tissue of patients with liver fibrosis and animal models of liver fibrosis, as well as PDGF-BB-induced activated HSC-T6. After downregulating the expression of ASIC1a, the degree of liver fibrosis is reduced and HSC activation was inhibited, the level of m6 A modification and miR-350 expression were also reduced. The results of dual luciferase reporter assay showed that miR-350 can bind to the target gene SPRY2 and inhibit its expression. We also found that METTL3 can regulate the extent of m6 A modification of pri-miR-350 by binding to DGCR8. In addition, silencing or blocking the expression of ASIC1a can reduce the expression of PI3K/AKT and ERK signaling pathway-related proteins in activated HSCs. Taken together, we demonstrated that ASIC1a regulates the processing of miR-350 through METTL3-dependent m6 A modification, and mature miR-350 targets SPRY2 and further promotes liver fibrosis through the PI3K/KT and ERK pathways.
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Canales Iónicos Sensibles al Ácido/metabolismo , Adenosina/análogos & derivados , Cirrosis Hepática/metabolismo , Proteínas de la Membrana/genética , MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Canales Iónicos Sensibles al Ácido/genética , Adenosina/metabolismo , Animales , Línea Celular , Células Cultivadas , Humanos , Hígado/metabolismo , Cirrosis Hepática/genética , Sistema de Señalización de MAP Quinasas , Masculino , Proteínas de la Membrana/metabolismo , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic ß-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance. METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing. RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2. CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.
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Sistemas CRISPR-Cas , Glucosa/metabolismo , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Gotas Lipídicas/metabolismo , Lipogénesis , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Perfilación de la Expresión Génica , Células Hep G2 , Hepatocitos/citología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Fosforilación , Transducción de SeñalRESUMEN
Multiple myeloma (MM) is an incurable hematological malignancy characterized by abnormal infiltration of plasma cells in the bone marrow. MicroRNAs (miRNAs) have emerged as crucial regulators in human tumorigenesis and tumor progression. miR-27, a novel cancer-related miRNA, has been confirmed to be implicated in multiple types of human tumors; however, its biological role in MM remains largely unknown. The present study aimed to characterize the biological role of miR-27 in MM and elucidate the potential molecular mechanisms. Here we found that miR-27 was significantly up-regulated in MM samples compared with normal bone marrow samples from healthy donors. Moreover, the log-rank test and Kaplan-Meier survival analysis displayed that MM patients with high miR-27 expression experienced a significantly shorter overall survival than those with low miR-27 expression. In the current study, we transfected MM cells with miR-27 mimics or miR-27 inhibitor to manipulate its expression. Functional studies demonstrated that miR-27 overexpression promoted MM cell proliferation, facilitated cell cycle progression, and expedited cell migration and invasion; whereas miR-27 knockdown inhibited cell proliferation, induced cell cycle arrest, and slowed down cell motility. Mechanistic studies revealed that Sprouty homolog 2 (SPRY2) was a direct target of miR-27 and that rescuing SPRY2 expression reversed the promoting effects of miR-27 on MM cell proliferation, migration, and invasion. Besides, miR-27 ablation suppressed tumorigenecity of MM cells in mouse xenograft models. Collectively, our data indicate that miR-27 exerts its oncogenic functions in MM by targetting SPRY2 and that miR-27 may be used as a promising candidate target in MM treatment.
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Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Mieloma Múltiple/genética , Animales , Carcinogénesis/genética , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Mieloma Múltiple/diagnóstico , Pronóstico , Regulación hacia ArribaRESUMEN
BACKGROUND: Early invasion and metastasis are responsible for the dismal prognosis of pancreatic ductal adenocarcinoma (PDAC), and epithelial-to-mesenchymal transition (EMT) is recognized as a crucial biological progress in driving tumor invasion and metastasis. The transcription factor FOXO3a is inactivated in various types of solid cancers and the loss of FOXO3a is associated with EMT and tumor metastasis. In this study, we sought to explore whether SPRY2, a regulator of receptor tyrosine kinase (RTK) signaling, is involved in FOXO3a-mediated EMT and metastasis in PDAC. METHODS: Immunohistochemistry was performed in 130 paired PDAC tissues and paracarcinomatous pancreatic tissues. Cell proliferation and apoptosis were assessed by cell counting kit and flow cytometry, while cell migration and invasion were evaluated with wound healing and transwell assays. The changes in mRNA and protein levels were estimated by qRT-PCR and western blot. BALB/c nude mice xenograft model was established to evaluate tumorigenesis and metastasis in vivo. RESULTS: FOXO3a expression was remarkably reduced in PDAC tissues, and correlated with metastasis-associated clinicopathologic characteristics and poor prognosis in patients with PDAC. In addition to the promotion of proliferation and suppression of apoptosis, knockdown of FOXO3a or SPRY2 induced EMT and promoted the migration and invasion of PDAC cells via activation of the ß-catenin/TCF4 pathway. Moreover, silencing of SPRY2 reversed the suppressor effects induced by FOXO3a overexpression on EMT-associated migration and invasion of PDAC cells, while blockade of ß-catenin reversed the effects of SPRY2 loss. FOXO3a knockdown decreased SPRY2 protein stability, whereas SPRY2 knockdown enhanced ß-catenin protein stability. In vivo, FOXO3a knockdown promoted the tumorigenic ability and metastasis of PDAC cells. CONCLUSIONS: Our study suggests that knockdown of FOXO3a induces EMT and promotes metastasis of PDAC by activation of the ß-catenin/TCF4 pathway through SPRY2. Thus, FOXO3a may represent a candidate therapeutic target in PDAC.
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Carcinoma Ductal Pancreático/secundario , Transición Epitelial-Mesenquimal , Proteína Forkhead Box O3/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Pancreáticas/patología , Factor de Transcripción 4/metabolismo , beta Catenina/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Movimiento Celular , Proliferación Celular , Femenino , Estudios de Seguimiento , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pronóstico , Transducción de Señal , Tasa de Supervivencia , Factor de Transcripción 4/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , Neoplasias PancreáticasRESUMEN
Sprouty2 (Spry2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are both well-established regulators of receptor tyrosine kinase (RTK) signaling, and knockdown of Spry2 or PTEN enhances axon regeneration of dorsal root ganglia (DRG) neurons. The major role of Spry2 is the inhibition of the rat sarcoma RAS/extracellular signal-regulated kinase (ERK) pathway, whereas PTEN acts mainly as an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In non-neuronal cells, Spry2 increases the expression and activity of PTEN, and PTEN enhances the amount of Spry2 by the inhibition of the microRNA-21 (miR-21) that downregulates Spry2. Applying dissociated DRG neuron cultures from wild-type (WT) or Spry2 deficient mice, we demonstrate that PTEN protein was reduced after 72 h during rapid axonal outgrowth on the laminin substrate. Furthermore, PTEN protein was decreased in DRG cultures obtained from homozygous Spry2-/- knockout mice. Vice versa, Spry2 protein was reduced by PTEN siRNA in WT and heterozygous Spry2+/- neurons. Knockdown of PTEN in DRG cultures obtained from homozygous Spry2-/- knockout mice promoted axon elongation without increasing axonal branching. Activation of Akt, but not ERK, was stronger in response to PTEN knockdown in homozygous Spry2-/- DRG neurons than in WT neurons. Together, our study confirms the important role of the signaling modulators Spry2 and PTEN in axon growth of adult DRG neurons. Both function as endogenous inhibitors of neuronal growth factor signaling and their simultaneous knockdown promotes axon elongation more efficiently than the single knockdown of each inhibitor. Furthermore, Spry2 and PTEN are reciprocally downregulated in adult DRG neuron cultures. Axon growth is influenced by multiple factors and our results demonstrate that the endogenous inhibitors of axon growth, Spry2 and PTEN, are co-regulated in adult DRG neuron cultures. Together, our data demonstrate that combined approaches may be more useful to improve nerve regeneration than targeting one single inhibitor of axon growth.
RESUMEN
AIM: Bile acid biosynthesis is strictly regulated under physiological conditions. The expression of fibroblast growth factor (FGF) 19 is induced when bile acids bind to the farnesoid X receptor in the intestinal epithelium. Fibroblast growth factor 19 is then transported by the portal flow, causing transcriptional inhibition of cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1), a key enzyme in bile acid biosynthesis, through the extracellular signal-regulated kinase (ERK) pathway. However, the regulatory mechanisms of these signaling pathways in hepatocytes under chronic cholestasis remain unclear. We investigated the regulation of these signaling pathways in patients with biliary atresia (BA). METHODS: We analyzed the regulation of molecules in these signaling pathways using liver and serum samples from eight BA children and four non-cholestatic disease controls. RESULTS: CYP7A1 mRNA expression was not inhibited in BA microdissected hepatocyte-enriched tissue (HET) despite high serum bile acid concentrations. The FGF19 protein was synthesized in BA HET, and its serum concentration was elevated. Fibroblast growth factor receptor 4 was phosphorylated in BA livers. However, ERK phosphorylation was significantly reduced. We examined SPRY2 expression to determine how the ERK pathway was inactivated downstream of the FGF receptor; the expression was significantly increased in BA HET. CONCLUSIONS: This is the first study to measure the CYP7A1 mRNA levels in human BA HET. Fibroblast growth factor 19 was increased in BA hepatocytes. By focusing on its regulation in hepatocytes, we showed that the FGF19 pathway did not suppress bile acid synthesis, probably due to an altered mechanism involving upregulated SPRY2 in BA patients.
RESUMEN
Prostate cancer (PC) is the most common cancer in men; however, limited effect is obtained due to the therapy resistance. CASC2 acts as a tumor suppressor in human malignancies serving as a ceRNA for miRNAs; Sprouty2 (SPRY2), a key antagonist of RTK signaling, also serves as a tumor suppressor. Herein, CASC2 and SPRY2 expression was down-regulated in PC tissues and cell lines; the overexpression of CASC2 and SPRY2 could suppress PC cell proliferation, promote PC cell apoptosis, and enhance the sensitivity of PC cells to docetaxel. CASC2 positively regulated SPRY2 expression and inhibited downstream extracellular regulated protein kinases (ERK) signaling activation through SPRY2. By using online tools, miR-183 might be a direct target of CASC2, and might simultaneously bind to the 3'UTR of SPRY2. The direct binding between CASC2, miR-183 and SPRY2 was then validated; miR-183 inhibition enhanced the cytotoxicity of docetaxel on PC cells, which could be partially attenuated by SPRY2 knockdown. In summary, CASC2 competes with SPRY2 for miR-183 binding to rescue the expression of SPRY2 in PC cells, thus enhancing the sensitivity of PC cells to docetaxel through SPRY2 downstream ERK signaling pathway; CASC2 and SPRY2 might be novel adjuvants for docetaxel-based chemotherapy for PC.
Asunto(s)
Antineoplásicos/farmacología , Docetaxel/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , MicroARNs/genética , Neoplasias de la Próstata/patología , ARN Largo no Codificante/genética , ARN/genética , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Neoplasias de la Próstata/genéticaRESUMEN
Background: Studies have shown that microRNAs are closely related to the occurrence and development of tumors. As one of the most common carcinogenic microRNAs, the specific role and mechanism of miR-21 in the proliferation and migration of pancreatic cancer cells have not been fully elucidated. Aims: To investigate the effect and specific molecular mechanism of miR-21 on proliferation and migration of pancreatic cancer cells. Methods: PANC-1 and MIA PaCa-2 cells were transfected with lentiviral vectors to construct stable cell lines with overexpression and knockdown of miR-21, respectively. The transfection efficiency was verified by qRT-PCR. CCK-8 assay was used to detect cell proliferation, scratch test was used to detect cell migration ability. The expressions of Spry2 mRNA and protein were determined by qRT-PCR and Western blotting, respectively, and luciferase report assay was used to detect the regulatory effect of miR-21 on Spry2. Results: Overexpression of miR-21 significantly promoted PANC-1 cells proliferation and migration (P<0.05). Knockdown of miR-21 significantly inhibited MIA PaCa-2 cells proliferation and migration (P<0.05). Compared with controls, expressions of Spry2 mRNA and protein were significantly decreased after up-regulation of miR-21 (P<0.05), however, expressions of Spry2 mRNA and protein were significantly increased after down-regulation of miR-21 (P<0.05). Overexpression of miR-21 could inhibit luciferase activity of plasmids containing wild-type Spry2 3'-UTR sequence (P<0.05), and knockdown of miR-21 could enhance luciferase activity (P<0.05). Spry2 could reverse miR-21-mediated cell proliferation and migration (P<0.05). Conclusions: MiR-21 can affect biological function of pancreatic cancer cells by targeted regulating the expression of Spry2.
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Deregulation of key signaling pathways is one of the primary phenomena in carcinogenesis. DAB2IP and SPRY2 are regulatory elements, which act as feedback inhibitors of receptor tyrosine kinases signaling in mitogen-activated protein kinase pathway. These elements have also been implicated in the pathophysiology of cancer. Therefore, this study is aimed to investigate the expression of all known splice variants of DAB2IP and SPRY2 in prostate tissue. Fresh Prostate tissue samples (50 prostate cancer/ matched normal tissue and 30 BPH) were collected and total RNA was extracted followed by cDNA synthesis. The expression of DAB2IP and SPRY2 transcript variants were evaluated using RT-PCR and quantitative Real-time PCR. The results indicated significant down-regulation of DAB2IP transcript variant 1 in cancerous tissues compared to paired normal tissues (P = 0.001) as well as SPRY2 transcript variant 2 in cancerous tissues in comparison with the normal counterparts and BPH (P = 0.008 and P = 0.025, respectively). In addition, there was a significant negative correlation between DAB2IP.1 and SPRY2.2 expression with PSA levels in prostate cancer (P = 0.039 ρ =-0.24 and P = 0.045 ρ =-0.3, respectively). Interestingly, the down-regulation of DAB2IP.1 mRNA and SPRY2.2 mRNA was positively correlated in tumor samples (P = 0.002 ρ = 0.434). For the first time, this experiment highlights the deregulation of DAB2IP and SPRY2 transcript variants in human prostate cancer. The present study confirms and extends the previous reports through indicating transcript-specific down-regulation and significant association of DAB2IP and SPRY2 in prostate tumorigenesis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Anciano , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
BACKGROUND/AIMS: Gastric cancer (GC) is one of the most prevalent digestive malignancies. MicroRNAs (miRNAs) are involved in multiple cellular processes, including oncogenesis, and miR-592 itself participates in many malignancies; however, its role in GC remains unknown. In this study, we investigated the expression and molecular mechanisms of miR-592 in GC. METHODS: Quantitative real-time PCR and immunohistochemistry were performed to determine the expression of miR-592 and its putative targets in human tissues and cell lines. Proliferation, migration, and invasion were evaluated by Cell Counting Kit-8, population doubling time, colony formation, Transwell, and wound-healing assays in transfected GC cells in vitro. A dual-luciferase reporter assay was used to determine whether miR-592 could directly bind its target. A tumorigenesis assay was used to study whether miR-592 affected GC growth in vivo. Proteins involved in signaling pathways and the epithelial-mesenchymal transition (EMT) were detected with western blot. RESULTS: The ectopic expression of miR-592 promoted GC proliferation, migration, and invasion in vitro and facilitated tumorigenesis in vivo. Spry2 was a direct target of miR-592 and Spry2 overexpression partially counteracted the effects of miR-592. miR-592 induced the EMT and promoted its progression in GC via the PI3K/AKT and MAPK/ERK signaling pathways by inhibiting Spry2. CONCLUSIONS: Overexpression of miR-592 promotes GC proliferation, migration, and invasion and induces the EMT via the PI3K/AKT and MAPK/ERK signaling pathways by inhibiting Spry2, suggesting a potential therapeutic target for GC.