RESUMEN
Cartilage generation and degradation are controlled by miRNAs. Our previous study showed miR-23a-3p was downregulated during chondrogenic differentiation in chondrogenic human adipose-derived mesenchymal stem cells (hADSCs). In the present study, we explored the function of miR-23a-3p in chondrogenesis differentiation. The role of miR-23a-3p in chondrogenic differentiation potential of hADSCs was assessed by Alcian blue staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. We show that miR-23a-3p suppressed the chondrogenic differentiation of hADSCs. LncRNA SNHG5 interacted with miR-23a-3p, and suppression or overexpression of SNHG5 correlates with inhibition and promotion of hADSC chondrogenic differentiation, respectively. We have determined that SNHG5 can sponge miR-23a-3p to regulate the expression of SOX6/SOX5, transcription factors that play essential roles in chondrocyte differentiation. Furthermore, the overexpression of SNHG5 activates the JNK/MAPK/ERK pathway. In conclusion, miR-23a-3p regulated by lncRNA SNHG5 suppresses the chondrogenic differentiation of human adipose-derived stem cells via targeting SOX6/SOX5.
Asunto(s)
Diferenciación Celular/genética , Condrogénesis/genética , Regulación de la Expresión Génica , Células Madre Mesenquimatosas/citología , MicroARNs/genética , ARN Largo no Codificante/metabolismo , Factores de Transcripción SOXD/metabolismo , Secuencia de Bases , Humanos , Sistema de Señalización de MAP Quinasas , Células Madre Mesenquimatosas/metabolismo , MicroARNs/metabolismo , ARN Largo no Codificante/genéticaRESUMEN
This study employed transforming growth factor beta 3 (TGF-ß3) induction of the C3H10T1/2 mesenchymal stem cell (MSC) line to construct an in vitro chondrogenic differentiation model for MSCs. A C3H10T1/2 MSC cell line was cultured, amplified, and the seventh generation of cells was centrifuged to construct pellets, which were divided into a non-induced group and an induced group (treated with TGF-ß3, vitamin C, dexamethasone, and ITS). Specimens were taken after 7, 14, 21, and 28 days under non-induced and induced culture to compare these two groups by Alcian Blue staining, collagen type II immunohistochemical staining, and transmission election microscopy (TEM) on days 21 and 42. Cell pellets in the non-induced group were smaller than those in the induced group on days 7, 14, 21, and 28 with the pellet morphology of the induced group being more regular and nearly spherical. Alcian blue staining in the induced group was consistently stronger than that in non-induced group across all time points, and type II collagen immunohistochemical IOD values were significantly higher in the induced group over the non-induced group across all time points. On days 21 and 42, TEM revealed that the induced group displayed greater karyokinesis and a higher euchromatin ratio compared to the non-induced group. This specially constructed pellet model treated with TGF-ß3-containing chondrogenic medium can effectively promote chondrogenic differentiation of C3H10T1/2 MSC cells in vitro. This in vitro pellet model should be of value in providing a preliminary cell model reference for further studies of the mechanism of chondroblast differentiation of stem cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Condrogénesis , Células Madre Mesenquimatosas/citología , Modelos Biológicos , Células Madre Embrionarias de Ratones/citología , Animales , Fusión Celular , Línea Celular , Forma de la Célula , Colágeno Tipo II/metabolismo , Inmunohistoquímica , Células Madre Mesenquimatosas/ultraestructura , Ratones , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
Chronic diabetic wounds are a severe complication of diabetes, often leading to high treatment costs and high amputation rates. Numerous studies have revealed that nitric oxide (NO) therapy is a promising option because it favours wound revascularization. Here, base-paired injectable adhesive hydrogels (CAT) were prepared using adenine- and thymine-modified chitosan (CSA and CST). By further introducing S-nitrosoglutathione (GSNO) and binary l-arginine (bArg), we obtained a NO sustained-release hydrogel (CAT/bArg/GSON) that was more suitable for the treatment of chronic wounds. The results showed that the expression of HIF-1α and VEGF was upregulated in the CAT/bArg/GSON group, and improved blood vessel regeneration was observed, indicating an important role of NO. In addition, the research findings revealed that following treatment with the CAT/bArg/GSON hydrogel, the viability of Staphylococcus aureus and Escherichia coli decreased to 14 ± 2 % and 6 ± 1 %, respectively. Moreover, the wound microenvironment was improved, as evidenced by a 60 ± 1 % clearance of DPPH. In particular, histological examination and immunohistochemical staining results showed that wounds treated with CAT/bArg/GSNO exhibited denser neovascularization, faster epithelial tissue regeneration, and thicker collagen deposition. Overall, this study proposes an effective strategy to prepare injectable hydrogel dressings with dual NO donors. The functionality of CAT/bArg/GSON has been thoroughly demonstrated in research on chronic wound vascular regeneration, indicating that CAT/bArg/GSON could be a potential option for promoting chronic wound healing. STATEMENT OF SIGNIFICANCE: This article prepares a chitosan hydrogel utilizing the principle of complementary base pairing, which offers several advantages, including good adhesion, biocompatibility, and flow properties, making it a good material for wound dressings. Loaded GSNO and bArg can steadily release NO and l-arginine through the degradation of the gel. Then, the released l-arginine not only possesses antioxidant properties but can also continue to generate a small amount of NO under the action of NOS. This design achieves a sustained and stable supply of NO at the wound site, maximizing the angiogenesis-promoting and antibacterial effects of NO. More neovascularization and abundant collagen were observed in the regenerated tissues. This study provides an effective repair hydrogel material for diabetic wound.
Asunto(s)
Quitosano , Diabetes Mellitus , Humanos , Hidrogeles/farmacología , Hidrogeles/química , Donantes de Óxido Nítrico/farmacología , Adhesivos/farmacología , Quitosano/farmacología , Quitosano/química , Angiogénesis , Cicatrización de Heridas , Colágeno/farmacología , Antibacterianos/farmacología , Arginina/farmacologíaRESUMEN
Excessive accumulation of exudate from wounds often causes infection and hinders skin regeneration. To handle wound exudate quickly and prevent infection, we developed an antibacterial Janus nanofibrous dressing with a unidirectional water-transport function. The dressing consists of a hydrophilic chitosan aerogel (CS-A) as the outer layer and a hydrophobic laurylated chitosan (La-CS) nanofibrous membrane as the inner layer. These dressings achieved excellent liquid absorption performance (2987.8⯱â¯123.5â¯%), air and moisture permeability (997.8⯱â¯23.1â¯g/m2/day) and mechanical strength (5.1⯱â¯2.6â¯MPa). This performance was obtained by adjusting the density of CS-A and the thickness of the La-CS membrane. Moreover, the dressing did not induce significant toxicity to cells and can prevent bacterial aggregation and infection at the wound site. Animal experiments showed that the dressing can shorten the inflammatory phase, enhance blood vessel generation, and accelerate collagen deposition, thus promoting wound healing. Overall, these results suggest that this Janus dressing is a promising material for clinical wound care.
Asunto(s)
Antibacterianos , Vendajes , Quitosano , Nanofibras , Agua , Cicatrización de Heridas , Quitosano/química , Quitosano/farmacología , Cicatrización de Heridas/efectos de los fármacos , Nanofibras/química , Antibacterianos/farmacología , Antibacterianos/química , Animales , Agua/química , Ratones , Interacciones Hidrofóbicas e Hidrofílicas , Permeabilidad , Ratas , Staphylococcus aureus/efectos de los fármacos , MasculinoRESUMEN
Three-dimensional grafts/scaffolds with hierarchically biomimetic features from nano to macro scale and high porosity are required for bone tissue engineering. In this study, biomimetic organic/inorganic composite scaffolds with high porosity (78.7 ± 3.2%) and features from nano (nano apatite coatings) to macro (macro pores and hollow channels) scale were fabricated based on highly concentrated alginate/GelMA bioinks via co-axial 3D printing and in situ mineralization under mild conditions. Nano apatites were coated on both inner and outer surfaces of the hollow fiber scaffolds, homogeneously. Proteins were directly loaded in the bioinks achieving sustained release from the scaffolds over 28 days. The in vitro cell experiments showed that the scaffolds with good biocompatibility could support cells adhesion and proliferation. The nano apatite coatings presented remarkable osteogenic capability. The in vivo study indicated that the hollow fiber scaffolds with biomimetic nano apatite coatings showed the capability to enhance bone formation after 12 weeks of implantation. In conclusion, the prepared biomimetic organic/inorganic scaffolds with homogeneous nano apatite coatings and hollow channels structures might be potential candidates for bone tissue engineering.
Asunto(s)
Apatitas , Ingeniería de Tejidos , Alginatos/química , Porosidad , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Mounting evidence demonstrates uncontrolled endoplasmic reticulum (ER) stress responses can activate the inflammasome, which generally results in endothelial dysfunction, a major pathogenetic factor of chronic inflammatory diseases such as atherosclerosis. Salvianolic acid B (SalB), produced by Radix Salviae, exerts antioxidative and anti-inflammatory activities in multiple cell types. However, SalB's effects on ER stress-related inflammasome and endothelial dysfunction remain unknown. Here, we showed SalB substantially abrogated ER stress-induced cell death and reduction in capillary tube formation, with declined intracellular reactive oxygen species (ROS) amounts and restored mitochondrial membrane potential (MMP), as well as increased expression of HO-1 and SOD2 in bone marrow-derived endothelial progenitor cells (BM-EPCs). ER stress suppression by CHOP or caspase-4 siRNA transfection attenuated the protective effect of SalB. Additionally, SalB alleviated ER stress-mediated pyroptotic cell death via the suppression of TXNIP/NLRP3 inflammasome, as evidenced by reduced cleavage of caspase-1 and interleukin- (IL-) 1ß and IL-18 secretion levels. Furthermore, this study provided a mechanistic basis that AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 signaling pathway modulation by SalB substantially prevented BM-EPCs damage associated with ER stress by decreasing intracellular ROS amounts and inducing NLRP3-dependent pyroptosis. In summary, our findings identify that ER stress triggered mitochondrial ROS release and NLRP3 generation in BM-EPCs, while SalB inhibits NLRP3 inflammasome-mediated pyroptotic cell death by regulating the AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 pathways. The current findings reveal SalB as a potential new candidate for the treatment of atherosclerotic heart disease.
Asunto(s)
Células Progenitoras Endoteliales , Inflamasomas , Proteínas Quinasas Activadas por AMP/metabolismo , Benzofuranos , Proteínas de Ciclo Celular/metabolismo , Células Progenitoras Endoteliales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis , Transducción de Señal , Sindecano-4/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Perillaldehyde (PAH), one of the active ingredients of the traditional Chinese medicine (TCM) plant Perilla frutescens, is widely used and exerts crucial anti-cancer activities. The aim of current study is to illustrate the potential mechanisms of PAH-mediated regulation of bone metastasis and osteoclastogenesis in prostate cancer (PCa) cell lines. Effects of PAH on proliferation, invasion and migration of PC-3 cells were assessed with the Cell Counting Kit-8 (CCK-8) assay and Transwell assays, respectively. Effects of PAH on stem cell characteristics of PC-3 cells were evaluated by cell-matrix adhesion assay, colony formation assay, spheroid formation assay, as well as western blot . The anti-metastasis and anti-osteoclastogenesis activity of PAH in RAW264.7 cells was examined by osteoclast differentiation assay and western blot. The protein levels of CD133 and CD44 in PC-3 cells and the activity of nuclear factor kappa B (NF-κB) signaling pathway in RAW264.7 cells were measured by western blot. PAH suppressed proliferation, invasion and migration of PC-3 cells, prevented stem cell characteristics including cell-matrix adhesion, colony formation, spheroid formation as well as CD133 and CD44 expression. PAH inhibited bone metastasis and osteoclastogenesis via repressing the activation of NF-κB pathway as well as (RANKL) - and cancer cell-induced osteoclastogenesis in PCa cells. These findings suggested the potential therapeutic effects of PAH on the metastasis of patients with PCa.
Asunto(s)
Neoplasias Óseas/prevención & control , Monoterpenos/farmacología , Proteínas de Neoplasias/metabolismo , Osteoclastos/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Ligando RANK/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Células PC-3 , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células RAW 264.7RESUMEN
Bone metastasis is a significant cause of morbidity and mortality in patients with prostate cancer (PCa). This study is aimed at illustrating the mechanism of sweroside-mediated regulation in bone metastasis in PCa cells. Owing to the limitations of antitumor drugs in terms of their physical and chemical properties, making them into nanomaterials can effectively improve drug stability and bioavailability. Apoptosis was assessed with flow cytometry using the annexin V/propidium iodide binding assay; proteins, including p53, P21, Bcl-2, and Bax; and induction of intracellular reactive oxygen species (ROS). Using colony formation assay, sphere formation assay, and the expression changes in CD133 and CD44, stem cell characteristics were assessed. Epithelial-mesenchymal transition (EMT) activity was accessed by levels of the expression changes of EMT-related markers, vimentin and E-cadherin. Wnt/ß-catenin signaling pathway was examined to detect the levels of the expression changes of snail and ß-catenin. PC-3 cells were treated with lithium chloride (LiCl), which is an agonist of Wnt/ß-catenin signaling, and the levels of CD133, CD44, vimentin, E-cadherin, snail, and ß-catenin were detected. T-cell factor/lymphocyte enhancer factor (TCF/LEF) activity in cells overexpressing ß-catenin was used to detect the effects on ß-catenin transcription, and the expression of c-myc, Cyclin D1, Survivin, and MMP-7 were used to detect Wnt downstream target genes. Our results suggest that sweroside induces apoptosis and intracellular ROS; upregulates apoptotic proteins; and suppresses proliferation, invasion, and migration, preventing stem cell characteristics, including sphere formation, colony formation, and CD133 and CD44 expressions. Furthermore, sweroside nanoparticles exerts inhibitory effects on ß-catenin transcription by suppressing TTCF/LEF activity in cells overexpressing ß-catenin and downregulation of the expression of Wnt downstream target genes, including c-myc, Cyclin D1, Survivin, and MMP-7. The potential therapeutic effect of sweroside nanoparticles on bone metastatis of PCa was suggested, by these findings.
Asunto(s)
Nanopartículas , Neoplasias de la Próstata , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Humanos , Glucósidos Iridoides , Masculino , Células PC-3 , Neoplasias de la Próstata/tratamiento farmacológico , Vía de Señalización WntRESUMEN
INTRODUCTION: Circ_HECW2 plays a key role in lipopolysaccharide (LPS)-induced signal transduction, which is critical in osteoarthritis (OA). Thus, we analyzed the role of Circ_HECW2 in osteoarthritis. METHODS: The expression of Circ_HECW2 and miR-93 was examined using reverse-transcription polymerase chain reaction. Cell apoptosis was evaluated using Annexin V-FITC Apoptosis Detection Kit. RESULTS: Circ_HECW2 and miR-93 were inversely correlated, with Circ_HECW2 upregulated and miR-93 downregulated in OA and LPS-induced chondrocytes. Circ_HECW2 overexpression inhibited miR-93 expression and increased methylation of miR-93 coding gene. Cell apoptosis analysis showed that Circ_HECW2 overexpression increased LPS-induced chondrocyte apoptosis, while MiR-93 overexpression reversed the effects of Circ_HECW2 on chondrocyte apoptosis. CONCLUSION: In summary, our data revealed that the Circ_HECW2 is highly expressed in OA and might inhibit miR-93 expression through methylation to affect LPS-induced chondrocyte apoptosis.
Asunto(s)
Condrocitos , MicroARNs , Apoptosis , Condrocitos/metabolismo , Lipopolisacáridos , Metilación , MicroARNs/genéticaRESUMEN
miR-532-3p is a widely documented microRNA (miRNA) involved in multifaceted processes of cancer tumorigenesis and metastasis. However, the clinical significance and biological functions of miR-532-3p in bone metastasis of prostate cancer (PCa) remain largely unknown. Herein, we report that miR-532-3p was downregulated in PCa tissues with bone metastasis, and downexpression of miR-532-3p was significantly associated with Gleason grade and serum prostate-specific antigen (PSA) levels and predicted poor bone metastasis-free survival in PCa patients. Upregulating miR-532-3p inhibited invasion and migration abilities of PCa cells in vitro, while silencing miR-532-3p yielded an opposite effect on invasion and migration abilities of PCa cells. Importantly, upregulating miR-532-3p repressed bone metastasis of PCa cells in vivo. Our results further demonstrated that overexpression of miR-532-3p inhibited activation of nuclear facto κB (NF-κB) signaling via simultaneously targeting tumor necrosis factor receptor-associated factor 1 (TRAF1), TRAF2, and TRAF4, which further promoted invasion, migration, and bone metastasis of PCa cells. Therefore, our findings reveal a novel mechanism contributing to the sustained activity of NF-κB signaling underlying the bone metastasis of PCa.
RESUMEN
The prime issue derived from prostate cancer (PCa) is its high prevalence to metastasize to bone. MicroRNA-204-5p (miR-204-5p) has been reported to be involved in the development and metastasis in a variety of cancers. However, the clinical significance and biological functions of miR-204-5p in bone metastasis of PCa are still not reported yet. In this study, we find that miR-204-5p expression is reduced in PCa tissues and serum sample with bone metastasis compared with that in PCa tissues and serum sample without bone metastasis, which is associated with advanced clinicopathological characteristics and poor bone metastasis-free survival in PCa patients. Moreover, upregulation of miR-204-5p inhibits the migration and invasion of PCa cells in vitro, and importantly, upregulating miR-204-5p represses bone metastasis of PCa cells in vivo. Our results further demonstrated that miR-204-5p suppresses invasion, migration, and bone metastasis of PCa cells via inactivating nuclear factor κB (NF-κB) signaling by simultaneously targeting TRAF1, TAB3, and MAP3K3. In clinical PCa samples, miR-204-5p expression negatively correlates with TRAF1, TAB3, and MAP3K3 expression and NF-κB signaling activity. Therefore, our findings reveal a new mechanism underpinning the bone metastasis of PCa, as well as provide evidence that miR-204-5p might serve as a novel serum biomarker in bone metastasis of PCa. This study identifies a novel functional role of miR-204-5p in bone metastasis of prostate cancer and supports the potential clinical value of miR-204-5p as a serum biomarker in bone metastasis of PCa.
RESUMEN
A number of studies have reported that aberrant expression of microRNAs (miRNAs) closely correlates with the bone metastasis of prostate cancer (PCa). However, clinical significance and functional roles of both strands of a single miRNA in bone metastasis of PCa remain undefined. Here, we reported that miR-582-3p and miR-582-5p expression were simultaneously reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Downexpression of miR-582-3p and miR-582-5p strongly and positively correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-582-3p and miR-582-5p inhibited invasion and migration abilities of PCa cells in vitro, as well as repressed bone metastasis in vivo. Our results further revealed that miR-582-3p and miR-582-5p attenuated bone metastasis of PCa via inhibiting transforming growth factor ß (TGF-ß) signaling by simultaneously targeting several components of TGF-ß signaling, including SMAD2, SMAD4, TGF-ß receptor I (TGFBRI), and TGFBRII. Moreover, deletion contributes to miR-582-3p and miR-582-5p downexpression in PCa tissues. Finally, clinical negative correlations of miR-582-3p and miR-582-5p with SMAD2, SMAD4, TGFBRI, and TGFBRII were demonstrated in PCa tissues. Thus, our findings explore a novel tumor-suppressive miRNA with its both strands implicated in bone metastasis of PCa, suggesting its potential therapeutic value in treatment of PCa bone metastasis.
RESUMEN
Osteosarcoma is a kind of high-risk sarcoma of the skeleton typically observed in people under 25 years old. Currently, radiotherapy is widely applied in cancer treatment. However, osteosarcoma is radioresistant and accordingly new, more effective radiosensitizers are needed. miRNAs have been reported to play an important role in osteosarcoma radiosensitivity. We examined the modulating effect of miR3283p in vivo and in vitro. miR3283p was downregulated in HOS2R cells. The overexpression of miR3283p enhanced the radiosensitivity of osteosarcoma cells. miR3283p inhibited proliferation and promoted apoptosis in osteosarcoma cells under radiation conditions. In cells overexpressing miR3283p, H2AX expression was downregulated. We found that miR3283p targets H2AX and inhibits its expression. It was concluded, that miR3283p enhances the radiosensitization of osteosarcoma following X-ray irradiation, and determined that it directly targets H2AX to regulate radiosensitization.
Asunto(s)
Neoplasias Óseas/radioterapia , Histonas/genética , MicroARNs/genética , Osteosarcoma/radioterapia , Tolerancia a Radiación , Regiones no Traducidas 3' , Animales , Apoptosis , Neoplasias Óseas/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Osteosarcoma/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Constitutive activation of TGFß signaling pathway is a well-documented mechanism responsible for the bone metastasis of prostate cancer (PCa). MicroRNAs (miRNAs) have been reported to be crucial for the activation of TGFß signaling via targeting downstream components of TGFß signaling pathway. Here, we report that miR19a3p is downregulated in bone metastatic PCa tissues and cells. Upregulation of miR19a3p suppresses invasion, migration in vitro and inhibits bone metastasis in vivo in PCa cells. Conversely, silencing miR19a3p yields the opposite effect. Our results further demonstrate that miR19a3p inhibits invasion and migration abilities of PCa cells via targeting downstream effectors of TGFß signaling, SMAD2 and SMAD4, resulting in the inactivation of TGFß signaling. Therefore, our results uncover a novel mechanistic understanding of miR19a3p-induced suppressive role in bone metastasis of PCa, which will facilitate the development of effective cancer therapy methods against PCa.
Asunto(s)
Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Regulación hacia Abajo , MicroARNs/genética , Neoplasias de la Próstata/patología , Transducción de Señal , Regiones no Traducidas 3' , Anciano , Anciano de 80 o más Años , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , Invasividad Neoplásica , Trasplante de Neoplasias , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Bone metastasis is a leading cause of morbidity and mortality in advanced prostate cancer (PCa). Downexpression of miR-133a-3p has been found to contribute to the progression, recurrence and distant metastasis in PCa. However, clinical significance of miR-133a-3p in bone metastasis of PCa, and the biological role of miR-133a-3p and its molecular mechanisms underlying bone metastasis of PCa remain unclear. METHODS: miR-133a-3p expression was evaluated in 245 clinical PCa tissues by real-time PCR. Statistical analysis was performed to evaluate the clinical correlation between miR-133a-3p expression and clinicopathological features, and overall and bone metastasis-free survival in PCa patients. The biological roles of miR-133a-3p in the bone metastasis of PCa were investigated both in vitro and in vivo. Bioinformatics analysis, real-time PCR, western blot and luciferase reporter analysis were applied to demonstrate the relationship between miR-133a-3p and its potential targets. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the anti-tumor role of miR-133a-3p. Clinical correlation of miR-133a-3p with its targets was verified in human PCa tissues. RESULTS: miR-133a-3p expression is reduced in PCa tissues compared with the adjacent normal tissues and benign prostate lesion tissues, particularly in bone metastatic PCa tissues. Low expression of miR-133a-3p is significantly correlated with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients by statistical analysis. Moreover, upregulating miR-133a-3p inhibits cancer stem cell-like phenotypes in vitro and in vivo, as well as attenuates anoikis resistance in vitro in PCa cells. Importantly, administration of agomir-133a-3p greatly suppresses the incidence of PCa bone metastasis in vivo. Our results further demonstrate that miR-133a-3p suppresses bone metastasis of PCa via inhibiting PI3K/AKT signaling by directly targeting multiple cytokine receptors, including EGFR, FGFR1, IGF1R and MET. The negative clinical correlation of miR-133a-3p with EGFR, FGFR1, IGF1R, MET and PI3K/AKT signaling activity is determined in clinical PCa tissues. CONCLUSION: Our results unveil a novel mechanism by which miR-133a-3p inhibits bone metastasis of PCa, providing the evidence that miR-133a-3p may serve as a potential bone metastasis marker in PCa, and delivery of agomir-133a-3p may be an effective anti-bone metastasis therapeutic strategy in PCa.
Asunto(s)
Neoplasias Óseas/secundario , MicroARNs/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/complicaciones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Neoplasias Óseas/mortalidad , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , Análisis de Supervivencia , TransfecciónRESUMEN
MicroRNAs (miRs) are involved in several physiological processes, including chondrogenic differentiation, however, their expression and roles in the chondrogenic differentiation of human adiposederived stem cells (hADSCs) remain to be fully elucidated to date. Our previous study showed that miR13073p was significantly downregulated during chondrogenic differentiation by microarray and northern blot analysis. The present study aimed to investigate the effects of miR13073p on chondrogenic differentiation and the underlying mechanisms. First, the decreased expression of miR13073p was confirmed by reverse transcriptionquantitative polymerase chain reaction analysis. Subsequently, gain and lossoffunction of miR13073p experiments showed that the overexpression of miR13073p suppressed the deposition of cartilage matrix proteoglycans and decreased the expression of cartilagerelated markers, including sex determining region Ybox 9, collagen type II α1 chain and aggrecan, whereas the knockdown of miR13073p had the opposite effect. In addition, bone morphogenetic protein receptor type 2 (BMPR2) was identified as a target of miR13073p. Further mechanistic investigations showed that miR13073p attenuated the chondrogenic differentiation of hADSCs at least partly by inhibiting BMPR2mothers against decapentaplegic signaling pathways. In conclusion, the findings revealed that miR13073p inhibited the chondrogenic differentiation of hADSCs by targeting BMPR2 and its downstream signaling pathway, which may provide novel therapeutic clues for the treatment of cartilage injury.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Diferenciación Celular/genética , Condrogénesis/genética , MicroARNs/genética , Células Madre/citología , Células Madre/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Modelos Biológicos , Interferencia de ARN , Transducción de SeñalRESUMEN
High avidity of bone metastasis is an important characteristic in prostate cancer (PCa). Downexpression of miR-133b has been reported to be implicated in the development, progression and recurrence in PCa. However, clinical significance and biological roles of miR-133b in bone metastasis of PCa remain unclear. Here we report that miR-133b is downregulated in PCa tissues and further decreased in bone metastatic PCa tissues. Downexpression of miR-133b positively correlates with advanced clinicopathological characteristics and shorter bone metastasis-free survival in PCa patients. Upregulating miR-133b inhibits invasion, migration in vitro and bone metastasis in vivo in PCa cells. Mechanistically, we find that miR-133b suppresses activity of TGF-ß signaling via directly targeting TGF-ß receptor I and II, which further inhibits bone metastasis of PCa cells. Our results further reveal that overexpression of REST contributes to miR-133b downexpression via transcriptional repression in PCa tissues. Importantly, silencing miR-133b enhances invasion and migration abilities in vitro and bone metastasis ability in vivo in REST-silenced PCa cells. The clinical correlation of miR-133b with TGFBRI, TGFBRII, REST and TGF-ß signaling activity is verified in PCa tissues. Therefore, our results uncover a novel mechanism of miR-133b downexpression that REST transcriptionally inhibits miR-133b expression in PCa cells, and meanwhile support the notion that administration of miR-133b may serve as a rational regimen in the treatment of PCa bone metastasis.
Asunto(s)
Neoplasias Óseas/secundario , Regulación hacia Abajo/genética , MicroARNs/genética , Neoplasias de la Próstata/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
BACKGROUND: Clinically, prostate cancer (PCa) exhibits a high avidity to metastasize to bone. miR-141-3p is an extensively studied miRNA in cancers and downregulation of miR-141-3p has been widely reported to be involved in the progression and metastasis of several human cancer types. However, the clinical significance and biological roles of miR-141-3p in bone metastasis of PCa are still unclear. METHODS: miR-141-3p expression was examined in 89 non-bone metastatic and 52 bone metastatic PCa tissues by real-time PCR. Statistical analysis was performed to investigate the clinical correlation between miR-141-3p expression levels and clinicopathological characteristics in PCa patients. The biological roles of miR-141-3p in bone metastasis of PCa were evaluated both in vitro and a mouse intracardial model in vivo. Bioinformatics analysis, Western blot, luciferase reporter and miRNA immunoprecipitation assays were performed to explore and examine the relationship between miR-141-3p and its potential targets. Clinical correlation of miR-141-3p with its targets was examined in clinical PCa tissues. RESULTS: miR-141-3p expression is reduced in bone metastatic PCa tissues compared with non-bone metastatic PCa tissues. Low expression of miR-141-3p positively correlates with serum PSA levels, Gleason grade and bone metastasis status in PCa patients. Furthermore, upregulating miR-141-3p suppresses the EMT, invasion and migration of PCa cells in vitro. Conversely, silencing miR-141-3p yields an opposite effect. Importantly, upregulating miR-141-3p dramatically reduces bone metastasis of PC-3 cells in vivo. Our results further show that miR-141-3p inhibits the activation of NF-κB signaling via directly targeting tumor necrosis factor receptor-associated factor 5(TRAF5) and 6 (TRAF6), which further suppresses invasion, migration and bone metastasis of PCa cells. The clinical negative correlation of miR-141-3p expression with TRAF5, TRAF6 and NF-κB signaling activity is demonstrated in PCa tissues. CONCLUSION: Our findings unravel a novel mechanism underlying the bone metastasis of PCa, suggesting that miR-141-3p mimics might represent a potential therapeutic avenue for the treatment of PCa bone metastasis.
Asunto(s)
Neoplasias Óseas/secundario , MicroARNs/metabolismo , FN-kappa B/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Regulación hacia Abajo , Humanos , Masculino , MicroARNs/biosíntesis , MicroARNs/genética , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Transducción de SeñalRESUMEN
The present study aimed to investigate the mechanisms underlying microRNA (miRNA)-mediated regulation of chondrogenic differentiation. Mouse embryo-derived stem cells C3H10T1/2 were cultured and chondrogenic differentiation was induced using transforming growth factor-ß3 (TGF-ß3). In addition, miRNA expression profiles were detected via miRNA array analysis, and quantitative polymerase chain reaction was performed to verify the differentially expressed miRNAs. Furthermore, bioinformatics software was used to predict the putative targets and the prediction was validated by dual-luciferase reporter assays and western blot analysis. In addition, cell proliferation and glycosaminoglycans were measured by a direct cell count method and alcian blue staining, respectively. Compared with the control group, 86 miRNAs were identified as differentially expressed in TGF-ß3-induced cells and the expression levels of 28 miRNAs were increased while the remaining 58 miRNAs exhibited a decline in expression. Amongst the differentially expressed miRNAs, miR-30b expression was observed to have significantly decreased during chondrogenic differentiation. SOX9 is a target gene of miR-30b, and miR-30b inhibits SOX9 expression during chondrogenic differentiation. Furthermore, the alcian blue staining results demonstrated that miR-30b inhibited early chondrogenic differentiation. However, the data of the present study indicated that miR-30b had no influence on C3H10T1/2 cell line proliferation. In conclusion, miR-30b is a key negative regulator of TGF-ß3-induced C3H10T1/2 cell chondrogenic differentiation, which functions by directly targeting SOX9.