RESUMO
Developing strategies to enhance cartilage differentiation in mesenchymal stem cells and preserve the extracellular matrix is crucial for successful cartilage tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in maintaining the extracellular matrix and chondrocyte phenotype, thus serving as a key regulator in chondral tissue engineering strategies. Recent studies have shown that Ubiquitin C-terminal hydrolase L1 (UCHL1) is involved in the deubiquitylation of HIF-1α. However, the regulatory role of UCHL1 in chondrogenic differentiation has not been investigated. In the present study, we initially validated the promotive effect of UCHL1 expression on chondrogenesis in adipose-derived stem cells (ADSCs). Subsequently, a hybrid baculovirus system was designed and employed to utilize three CRISPR activation (CRISPRa) systems, employing dead Cas9 (dCas9) from three distinct bacterial sources to target UCHL1. Then UCHL1 and HIF-1α inhibitor and siRNA targeting SRY-box transcription factor 9 (SOX9) were used to block UCHL1, HIF-1α and SOX9, respectively. Cartilage differentiation and chondrogenesis were measured by qRT-PCR, immunofluorescence and histological staining. We observed that the CRISPRa system derived from Staphylococcus aureus exhibited superior efficiency in activating UCHL1 compared to the commonly used the CRISPRa system derived from Streptococcus pyogenes. Furthermore, the duration of activation was extended by utilizing the Cre/loxP-based hybrid baculovirus. Moreover, our findings show that UCHL1 enhances SOX9 expression by regulating the stability and localization of HIF-1α, which promotes cartilage production in ADSCs. These findings suggest that activating UCHL1 using the CRISPRa system holds significant potential for applications in cartilage regeneration.
Assuntos
Diferenciação Celular , Condrogênese , Subunidade alfa do Fator 1 Induzível por Hipóxia , Fatores de Transcrição SOX9 , Ubiquitina Tiolesterase , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Diferenciação Celular/genética , Condrogênese/genética , Animais , Humanos , Cartilagem/metabolismo , Condrócitos/metabolismo , Condrócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Sistemas CRISPR-Cas , CamundongosRESUMO
BACKGROUND: Diabetes mellitus (DM) and complications such as chronic kidney disease and cardiovascular symptoms pose a substantial public health burden. Increasing studies have shown that circular RNAs (circRNAs) regulate many gene expressions that are essential in diverse pathological and biological procedures. However, the roles of particular circRNAs in DM are unclear. METHODS: In the current investigation, endothelial progenitor cells (EPCs) were used to search for abnormal expression of circRNAs by using high-throughput sequencing under high glucose (HG) conditions. The regulatory mechanisms and targets were then studied through bioinformatics analysis, luciferase reporter analysis, angiogenic differentiation experiments, flow cytometry detection of apoptosis and RT-qPCR analysis. RESULTS: The circ-Astn1 expression in EPCs decreased after HG treatment. Overexpression or circ-Astn1 suppressed HG induced endothelial cell damage. MicroRNA (miR)-138-5p and SIRT5 were found to be the downstream targets of circ-Astn1 through luciferase reporter analysis. SIRT5 downregulation or miR-138-5p overexpression reversed circ-Astn1's protective effect against HG induced endothelial cell dysfunction, including apoptosis and abnormal vascular differentiation. Furthermore, circ-Astn1 overexpression promoted autophagy activation by increasing SIRT5 expression under HG conditions. Our findings suggest that circ-Astn1 mediated promotion of SIRT5 facilitates autophagy by sponging miR-138-5p. CONLUSION: Together, our findings show that the overexpression of circ-Astn1 suppresses HG induced endothelial cell damage by targeting miR-138-5p/SIRT5 axis.
RESUMO
m6A demethylase FTO is confirmed to be involved in pancreatic cancer progression. FTO regulates miRNA processing. To investigate the regulatory effect of FTO on miR-383-5p and its role in pancreatic cancer. The expression of miR-383-5p, ITGA3, and FTO was predicted using bioinformatic analysis in tissues and was measured using qPCR in cells. Cell biological functions were investigated using MTT assay, Transwell assay, sphere formation assay, and qPCR. The targeting relationship between miR-383-5p and ITGA3 was evaluated using the dual-luciferase reporter assay. The effect of FTO on miR-383-5p processing was evaluated using RIP and MeRIP assay. FTO expression was upregulated in pancreatic cancer and silencing of FTO promoted the processing of miR-383-5p in an m6A-dependent manner. m6A-modified miRNA processing was recognized by IGF2BP1. Downregulation of miR-383-5p reversed FTO knockdown-induced inhibition of cellular processes. The FTO/miR-383-5p/ITGA3 axis facilitated cell viability, metastasis, and stemness in pancreatic cancer.
RESUMO
Probiotic supplements were shown to improve cognitive function in Alzheimer's disease (AD) patients. However, it is still unclear whether this applies to older individuals with mild cognitive impairment (MCI). We aimed to explore the effects of probiotic supplementation on multiple neural behaviors in older adults with MCI. Forty-two MCI patients (age > 60 years) were randomly divided into two groups and consumed either probiotics (n=21) or placebo (n=21) for 12 weeks. Various scale scores, gut microbiota measures and serological indicators were recorded pre- and posttreatment. After 12 weeks of intervention, cognitive function and sleep quality were improved in the probiotic group compared with those in the control group, and the underlying mechanisms were associated with changes in the intestinal microbiota. In conclusion, our study demonstrated that probiotic treatment enhanced cognitive function and sleep quality in older MCI patients, thus providing important insights into the clinical prevention and treatment of MCI.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Probióticos , Humanos , Idoso , Disfunção Cognitiva/terapia , Cognição , Doença de Alzheimer/terapia , Probióticos/uso terapêutico , Probióticos/farmacologia , Suplementos NutricionaisRESUMO
New antifungals are urgently needed to combat invasive fungal infections, due to limited types of available antifungal drugs and frequently encountered side effects, as well as the quick emergence of drug-resistance. We previously developed amine-pendent poly(2-oxazoline)s (POXs) as synthetic mimics of host defense peptides (HDPs) to have antibacterial properties, but with poor antifungal activity. Hereby, we report the finding of short guanidinium-pendent POXs, inspired by cell-penetrating peptides, as synthetic mimics of HDPs to display potent antifungal activity, superior mammalian cells versus fungi selectivity, and strong therapeutic efficacy in treating local and systemic fungal infections. Moreover, the unique antifungal mechanism of fungal cell membrane penetration and organelle disruption explains the insusceptibility of POXs to antifungal resistance. The easy synthesis and structural diversity of POXs imply their potential as a class of promising antifungal agents.
Assuntos
Anti-Infecciosos , Micoses , Animais , Anti-Infecciosos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fungos , Guanidina/farmacologia , Mamíferos , Testes de Sensibilidade Microbiana , Micoses/tratamento farmacológico , OxazóisRESUMO
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, and no effective therapies have been found to prevent or cure AD to date. Berberine and curcumin are extracts from traditional Chinese herbs that have a long history of clinical benefits for AD. Here, using a transgenic AD mouse model, we found that the combined berberine and curcumin treatment had a much better effect on improving the cognitive function of mice than the single-drug treatment, suggesting synergic effects of the combined berberine and curcumin treatment. In addition, we found that the combined berberine and curcumin treatment had significant synergic effects on reducing soluble amyloid-ß-peptide(1-42) production. Furthermore, the combination treatment also had remarkable synergic effects on decreasing inflammatory responses and oxidative stress in both the cortex and hippocampus of AD mice. We also found that the combination treatment performed much better than the single drugs in reducing the APP and BACE1 levels and increasing AMPKα phosphorylation and cell autophagy, which might be the underlying mechanism of the synergic effects. Taken together, the result of this study reveal the synergic effects and potential underlying mechanisms of the combined berberine and curcumin treatment in improving the symptoms of AD in mice. This study sheds light on a new strategy for exploring new phytotherapies for AD and also emphasizes that more research should focus on the synergic effects of herbal drugs in the future.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Berberina/administração & dosagem , Encéfalo/efeitos dos fármacos , Cognição/efeitos dos fármacos , Curcumina/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/biossíntese , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/biossíntese , Encéfalo/metabolismo , Cognição/fisiologia , Sinergismo Farmacológico , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/biossínteseRESUMO
BACKGROUND: Increasing evidence has shown that circular RNAs (circRNAs) are involved tumourigenesis and metastasis of hepatocellular carcinoma (HCC); however, progression about its function in HCC is relatively slow. Here, we aimed to investigate whether plasma circRNAs could reflect the tumour-infiltrating lymphocytes (TILs) in HCC tumour tissues and serve as prognosis biomarker for HCC. METHODS: Tissue samples of patients with HCC were subjected to immunohistochemistry staining against CD8 to examine the TILs. Then, we investigated the expression profile of circRNAs by microarray between plasma of patients with HCC with high TILs and low TILs, and the differentially expressed circRNAs were validated with qRT-PCR. Statistical analysis was performed with SPSS software and GraphPad Prism. RESULTS: We have demonstrated that patients with HCC with high TILs exhibit a significant better overall survival, suggesting clinical outcome could be predicted by TILs. Global circRNA microarray between plasma of patients with HCC with high TILs and low TILs successfully identified six differentially expressed novel circRNAs. Among them, the expression of hsa_circ_0064428 was significantly reduced in patients with HCC with high TILs but increased in patients with low TILs. Moreover, hsa_circ_0064428 was negatively correlated with patient's survival, tumour size and metastasis. CONCLUSION: These findings together imply that hsa_circ_0064428 could be considered as a potential HCC prognosis biomarker. Future in-depth research is required to further illustrate the involvement of hsa_circ_0064428 in HCC tumourigenesis and metastasis.
Assuntos
Biomarcadores Tumorais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , RNA Circular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Estadiamento de Neoplasias , Prognóstico , Reprodutibilidade dos Testes , Análise de SobrevidaRESUMO
Diabetic foot ulcer (DFU) is one of diabetic complications, which is frequently present and tormented in diabetes mellitus. Most multipotent mesenchymal stromal cells (MSCs) are capable of immune evasion, providing an allogeneic, ready-to-use, cell product option for therapeutic applications. The beneficial effect of MSCs for the treatment of a variety of traumatic injuries, such as open wounds, has been extensively explored. In this study, a rat DFU model was used to simulate the pathophysiology of clinical patients and to investigate the localization of human umbilical cord mesenchymal stem cells (hUC-MSCs) after intravenous transplantation and its role in DFU healing, so as to evaluate the potential of hUC-MSCs in the treatment of DFU. The diabetic rat model was established by streptozotocin injection, which was used to create full-thickness foot dorsal skin wounds to mimic DFU by a 6-mm skin biopsy punch and a Westcott scissor. The hUC-MSCs were transplanted through femoral vein, and the ulcer cicatrization situation and the fate of hUC-MSCs were evaluated. Our data suggest that intravenously transplantated hUC-MSCs have the ability to migrate and locate to the wound tissue and are helpful to wound healing in DFU rats, partly by regulating inflammation, trans-differentiation and providing growth factors that promote angiogenesis, cell proliferation and collagen deposition. Herein, we demonstrate that hUC-MSC transplantation is able to accelerate DFU healing in rats and transplantation of exogenous stem cells may be a potential strategy for clinical application in DFUs.
Assuntos
Diabetes Mellitus Experimental , Pé Diabético , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Veias Umbilicais/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/terapia , Pé Diabético/metabolismo , Pé Diabético/patologia , Pé Diabético/terapia , Feminino , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/patologia , Ratos , Ratos Sprague-Dawley , Veias Umbilicais/patologiaRESUMO
BACKGROUND: Radiofrequency ablation (RFA) and antiarrhythmic drugs (AADs) are the main treatments used for atrial fibrillation (AF). In recent years, a number of articles comparing the 2 treatments have begun to emerge. Though, the influence of follow-up time in the meta-analysis was not considered in these articles. However, more recently, large-scale clinical trial articles have included follow-up with the patients up to 5 years after treatment. Therefore, the aim of this study was to assess the impact of variable follow-up times on the recurrence of AF by observing both the short-term and long-term efficacy and safety of catheter ablation and AADs for the treatment of AF. METHODS AND RESULTS: The primary investigators of eligible randomized controlled trials were invited to contribute standardized outcome data. Random effect summary estimates were calculated as standardized mean differences and odds ratios with 95% confidence intervals for continuous and binary outcomes. In this study, 9 randomized controlled trials (n = 1542 patients) were included. The rate of recurrence of AF with no limit on follow-up time, >12 months, >18 months, >24 months, >30 months, and approximately 36 months was compared. Furthermore, the gap between the RFA and AAD groups in the recurrence rate of AF was found to decrease inversely to follow-up time. When the follow-up time reached 24 months, the difference between RFA and AAD was relatively stable with an odds ratio of 0.45 (95% confidence interval: 0.32-0.62). Overall, RFA decreased adverse events in the remaining trials; however, AAD performed better in terms of safety and had fewer adverse events with RFA usually causing more serious complications. CONCLUSION: RFA is more advantageous in terms of recurrence rate of AF than drug therapy. In addition, the analysis suggests that this effect persists during long-term follow-up; however, these benefits appear to decrease with longer follow-up time. Finally, AAD performed better in terms of safety and had fewer adverse events.
Assuntos
Antiarrítmicos/uso terapêutico , Fibrilação Atrial/terapia , Ablação por Cateter , Frequência Cardíaca/efeitos dos fármacos , Potenciais de Ação , Idoso , Antiarrítmicos/efeitos adversos , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/mortalidade , Fibrilação Atrial/fisiopatologia , Ablação por Cateter/efeitos adversos , Ablação por Cateter/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Recidiva , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Diabetic ischemic ulcer is an intractable diabetic complication. Angiogenesis is a critical factor for wound healing in patients with diabetic foot wounds. Sustained gene delivery could be notably necessary in modulating gene expression in chronic ulcer healing and might be a promising approach for diabetic foot ulcers. In the present study, Sprague-Dawley rats were used to establish diabetic foot ulcer models by streptozotocin and skin biopsy punch. The plasmids expressing VEGF-A and PDGF-B were prepared and then incorporated with polylactic-co-glycolic acid (PLGA) nanospheres to upregulate genes expression. The aim of this study was to explore whether the engineered VEGF-A and PDGF-B based plasmid-loaded nanospheres could be upregulated in streptozotocin-induced diabetic rats and improve the wound healing. The cultured fibroblasts could be effectively transfected by means of nanosphere/plasmid in vitro. In vivo, the expression of VEGF-A and PDGF-B was significantly upregulated at full-thickness foot dorsal skin wounds and the area of ulceration was progressively and significantly reduced following treatment with nanosphere/plasmid. These results indicated that combined gene transfer of VEGF-A and PDGF-B could improve reparative processes in the wounded skin of diabetic rats and nanosphere may be a potential non-viral vector for gene therapy of the diabetic foot ulcer.
Assuntos
Pé Diabético/terapia , Úlcera do Pé/terapia , Proteínas Proto-Oncogênicas c-sis/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Diabetes Mellitus Experimental , Pé Diabético/genética , Pé Diabético/fisiopatologia , Modelos Animais de Doenças , Úlcera do Pé/genética , Úlcera do Pé/fisiopatologia , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Nanosferas/uso terapêutico , Plasmídeos/genética , Proteínas Proto-Oncogênicas c-sis/administração & dosagem , Ratos , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , CicatrizaçãoRESUMO
BACKGROUND & AIMS: Placement of an irradiation stent has been demonstrated to offer longer patency and survival than an uncovered self-expandable metallic stent (SEMS) in patients with unresectable malignant biliary obstruction (MBO). We aim to further assess the efficacy of an irradiation stent compared to an uncovered SEMS in those patients. METHODS: We performed a randomized, open-label trial of participants with unresectable MBO at 20 centers in China. A total of 328 participants were allocated in parallel to the irradiation stent group (ISG) or the uncovered SEMS group (USG). Endpoints included stent patency (primary), technical success, relief of jaundice, overall survival, and complications. RESULTS: The first quartile stent patency time (when 25% of the patients experienced stent restenosis) was 212â¯days for the ISG and 104â¯days for the USG. Irradiation stents were significantly associated with a decrease in the rate of stent restenosis (9% vs. 15% at 90â¯days; 16% vs. 27% at 180â¯days; 21% vs. 33% at 360â¯days; pâ¯=â¯0.010). Patients in the ISG obtained longer survival time (median 202â¯days vs. 140â¯days; pâ¯=â¯0.020). No significant results were observed in technical success rate (93% vs. 95%; pâ¯=â¯0.499), relief of jaundice (85% vs. 80%; pâ¯=â¯0.308), and the incidence of grade 3 and 4 complications (8.5% vs. 7.9%; pâ¯=â¯0.841). CONCLUSIONS: Insertion of irradiation stents instead of uncovered SEMS could improve patency and overall survival in patients with unresectable MBO. LAY SUMMARY: For patients with unresectable malignant biliary obstruction (MBO), placement of a self-expandable metallic stent (SEMS) is a recommended palliative modality to relieve pruritus, cholangitis, pain, and jaundice. However, restenosis is a main pitfall after stent placement. Data from this first multicenter randomized controlled trial showed that insertion of an irradiation stent provided longer patency and better survival than a conventional metal stent. ClinicalTrials.gov ID: NCT02001779.
Assuntos
Neoplasias do Sistema Biliar/complicações , Neoplasias do Sistema Biliar/terapia , Braquiterapia/métodos , Colestase/etiologia , Colestase/terapia , Stents , Idoso , Braquiterapia/efeitos adversos , Braquiterapia/instrumentação , China , Feminino , Humanos , Radioisótopos do Iodo/administração & dosagem , Masculino , Pessoa de Meia-Idade , Cuidados Paliativos/métodos , Stents Metálicos Autoexpansíveis/efeitos adversos , Stents/efeitos adversosRESUMO
The effects of long non-coding RNAs (lncRNAs) on cellular biological processes and even the tumorigenesis have been widely reported. Small nucleolar RNA host gene 6 (SNHG6) has been reported to participate in regulating biological behaviors of multiple types of cancers. Nevertheless, the functions of SNHG6 in osteosarcoma still remain to be uncovered. This study intended to determine the clinical significance and biological functions of SNHG6 in osteosarcoma. It was confirmed by qRT-PCR that SNHG6 was highly expressed in osteosarcoma tissues and cell lines. Highly expressed SNHG6 predicted poor survival rate and advanced clinical stage for osteosarcoma patients, according to Kaplan-Meier method and Cox regression analysis. Loss-of-function assays were performed to examine the effects of silenced SNHG6 on the progression of osteosarcoma, indicating that silenced SNHG6 suppressed cell proliferation through inducing cell cycle arrest in G0/G1 phase and causing cell apoptosis. In vitro assays exposed the potential oncogenic role of SNHG6 in osteosarcoma, further affirmed by in vivo nude mice assays. Mechanistic assays demonstrated that SNHG6 was negatively correlated with p21 and KLF2 in osteosarcoma. And biological functions of SNHG6 in osteosarcoma were realized through regulating p21 and KLF2. Collectively, SNHG6 was a new type of molecule involving in the progression of osteosarcoma.
Assuntos
Neoplasias Ósseas/diagnóstico , Inibidor de Quinase Dependente de Ciclina p21/genética , Fatores de Transcrição Kruppel-Like/genética , Osteossarcoma/diagnóstico , RNA Longo não Codificante/genética , Adulto , Animais , Apoptose/genética , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Nus , Osteossarcoma/genética , Prognóstico , Regulação para Cima , Adulto JovemRESUMO
INTRODUCTION: Leadless cardiac pacemaker (LCP) implantation using a transcatheter was recently developed to avoid pocket- and lead-related complications. Although a LCP has an active fixation mechanism using tines or a helix, LCP and lead dislodgement issues remain a major safety concern for patients. This article reviews the literature to determine the incidence of lead and LCP dislodgement. METHODS AND RESULTS: A total of 18 studies which included 17,321 patients undergoing conventional single- or dual-chamber pacemaker implantation and three studies which included 2,116 patients undergoing LCP device implantation were reviewed. The incidence of lead dislodgement ranged from 1% to 2.69% in individual studies with a mean of 1.63%, weighted mean of 1.71%, and median of 1.60 %. There was a relatively higher lead dislodgement rate between atrial and ventricular electrodes (odds ratio [OR], 3.56; 95% confidence interval [CI], 1.9-6.70; P = 0.6; I2 = 0%), and between magnetic resonance imaging conditional and conventional leads (OR, 2.79; 95% CI, 1.30-5.99; P = 0.16; I2 = 46%). The use of active fixation leads (OR, 1.06; 95% CI, 0.66-1.70; P = 0.29; I2 = 20%) showed no significant difference in dislodgement risk compared to passive fixation leads. The incidence of LCP device dislodgement was 0%, 0.13%, and 1% in three leadless pacemaker studies. CONCLUSIONS: The incidence rates of conventional pacemaker lead dislodgement vary in individual studies with an overall high incidence. Use of the currently available LCP systems appears to result in a lower rate of device dislodgement. This may reflect the effectiveness of this novel technology and the fixation design of LCP devices.
Assuntos
Falha de Equipamento , Marca-Passo Artificial/efeitos adversos , Desenho de Equipamento , Humanos , Incidência , Fatores de RiscoRESUMO
Diabetic nephropathy is a common kidney condition in patients with diabetes mellitus, which can result in renal failure. Pyroptosis, the process of pro-inflammatory programmed cell death, plays an important role in the pathogenesis of this disease. Long non-coding RNA MALAT1 has also been shown to be involved in diabetic nephropathy. Here, we investigated the role of MALAT1 and the microRNA miR-23c and its target gene ELAVL1 in renal tubular epithelial cells. Our data demonstrated that MALAT1 expression was substantially increased but miR-23c was decreased in streptozotocin-induced diabetic rats and in high-glucose-treated HK-2 cells. Downregulation of MALAT1 or upregulation the expression of miR-23c inhibited pyroptosis in HK-2 cells. In an effort to understand the signaling mechanisms underlying the pro-pyroptotic properties of MALAT1 and the anti-pyroptotic properties of miR-23c, we found that inhibiting the expression of MALAT1 downregulated the expression of ELAVL1, NLRP3, Caspase-1 and the pro-inflammatory cytokine IL-1ß. These findings were replicated by upregulation of miR-23c. Moreover, luciferase assays showed that miR-23c, as a target of MALAT1, directly repressed ELAVL1 expression and then decreased the expression of its downstream protein NLRP3. The expression of MALAT1 antagonized the effect of miR-23c on the downregulation of its target ELAVL1 and inhibited hyperglycemia-induced cell pyroptosis. This mechanism may contribute to a better understanding of diabetic nephropathy pathogenesis and facilitate the development of new therapeutic strategies for the treatment of this disease.
Assuntos
Nefropatias Diabéticas/metabolismo , Proteína Semelhante a ELAV 1/genética , MicroRNAs/genética , Piroptose , RNA Longo não Codificante/genética , Animais , Caspase 1/genética , Caspase 1/metabolismo , Linhagem Celular , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Proteína Semelhante a ELAV 1/metabolismo , Células Epiteliais/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The goal of this study was to characterize the properties of human CD34+ cells in culture and investigate the feasibility and efficacy of CD34+ transplantation in a mouse model of limb ischemia and in patients with no-option critical limb ischemia. METHODS: Human CD34+ cells isolated from peripheral blood and grown in culture for up to four passages stained positively for the surface markers CD34 and CD133 and showed high viability after cryopreservation and recovery. Seven days after surgery to induce limb ischemia, ischemic muscles of nude mice were injected with CD34+ cells. Two weeks later, mice were scored for extent of ischemic injury, and muscle tissue was collected for immunohistochemical analysis of vascular endothelial cells and RT-PCR analysis of cytokine expression. RESULTS: Injury scores of CD34+ -treated, but not control, mice were significantly different before and after transplantation. Vascular density and expression of VEGF and bFGF mRNAs were also significantly increased in the treated mice. Patients with severe lower extremity arterial ischemia were injected with their own CD34+ cells in the affected calf, foot, or toe. Significant improvements were observed in peak pain-free walking time, ankle-brachial index, and transcutaneous partial oxygen pressure. These findings demonstrate that growth of human CD34+ cells in vitro and cryopreservations are feasible. CONCLUSION: Such cells may provide a renewable source of stem cells for transplantation, which appears to be a feasible, safe, and effective treatment for patients with critical limb ischemia.
Assuntos
Antígenos CD34/química , Transplante de Medula Óssea , Isquemia/terapia , Adulto , Idoso , Animais , Células da Medula Óssea/química , Células da Medula Óssea/citologia , Células Cultivadas , Feminino , Membro Posterior/lesões , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Adulto JovemRESUMO
Endothelial malfunctions in patients with diabetes are known to result in vascular diseases, and endothelial progenitor cells (EPCs) are indispensable for the functional preservation of the vascular endothelium. MicroRNA-31 (miR-31) has been found to be able to modulate the differentiation of stem cells. However, it is still unclear how miR-31 functions in diabetic EPCs. The aim of this study was to investigate how miR-31 regulates diabetic EPC function. In the current study, miR-31 expression was compared between normal and diabetic EPCs. Satb2 was recognized as a functionally related target of miR-31 in EPCs according to computational prediction. We also explored the role of miR-31 in terms of its anti-apoptotic effects. A remarkable elevation in miR-31 expression was found in diabetic EPCs, and this elevated expression resulted in suppressed cell proliferation under high glucose. It was also found that miR-31 targets Satb2, leading to the anti-apoptotic effect and maintenance of the functions of EPCs. Furthermore, knockdown of Satb2 exhibited an inhibitory effect on proliferation and migration of EPCs in both healthy and diabetic subjects, which showed the same trend as miR-31 overexpression. Conversely, overexpression of Satb2 showed the opposite effect. Moreover, overexpression of Satb2 attenuated the miR-31-induced migration and colony-forming ability reduction and apoptosis induction of EPCs in both healthy and diabetic subjects. In diabetic EPCs, elevated glucose level was found to up-regulate miR-31 expression, which in turn enhanced the malfunction and death of EPCs. In conclusion, our results indicate that up-regulation of miR-31 may underlie endothelial dysfunction in diabetes by targeting Satb2.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Idoso , Apoptose , Movimento Celular , Proliferação de Células , Células Progenitoras Endoteliais/citologia , Feminino , Perfilação da Expressão Gênica , Glucose/química , Humanos , Masculino , Pessoa de Meia-Idade , TransfecçãoRESUMO
The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. Here, we show that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppresses Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promotes tissue growth when apoptosis is prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. Thus, our results have identified a novel molecular link between the Hippo and JNK pathways and implicated the essential role of the JNK pathway in Hippo signaling-related tumorigenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Discos Imaginais/embriologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Asas de Animais/embriologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Discos Imaginais/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Asas de Animais/citologia , Proteínas de Sinalização YAP , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Atherosclerosis is a disease resulting from impaired endothelial function, often caused by oxidant injury or inflammation. Endothelial progenitor cells (EPCs) play a critical role in repairing damaged endothelium and protecting against atherosclerosis. Quercitrin, a plant-derived flavonoid compound, displays antioxidant and anti-inflammatory activities. In this study, we showed that quercitrin treatment reduced the apoptosis of EPCs caused by oxidized low-density lipoprotein (ox-LDL) in a dose-dependent manner. Quercitrin improved tube formation, migration and adhesion of ox-LDL-treated EPCs. To determine the effect of quercitrin in vivo, EPCs treated with or without ox-LDL and quercitrin were locally injected into the ischemic hind limb muscle of nude mice. Those injected with EPCs treated with ox-LDL and quercitrin showed significantly increased local accumulation of EPCs, blood flow recovery and capillary density compared with the control and ox-LDL only groups. Furthermore, we showed that quercitrin enhanced autophagy and upregulated mitogen-activated protein kinase and ERK phosphorylation in a dose-dependent manner in vitro. Autophagy inhibitors, chloroquine and 3-methyladenine, abrogated quercitrin-enhanced autophagy caused by ox-LDL as evidenced by decreased numbers of branch points, migratory cells and adherent cells, and increased numbers of apoptotic cells. The ERK inhibitor PD98059 abrogated quercitrin-enhanced autophagy, as identified by decreased autophagosome formation and downregulated ERK phosphorylation. The inhibition of ERK did not affect the expression of Rac1, but enhanced phosphorylation of Akt. Quercitrin treatment also increased the expression of E-cadherin, and PD98059 abrogated the upregulation of E-cadherin induced by quercitrin. Our findings suggested that autophagy is a protective mechanism in EPCs exposed to oxidative damage. Quercitrin can promote autophagy through the activation of ERK and the ERK signaling pathway is therefore thought to play a pivotal role in mediating the protective effects on EPCs.
Assuntos
Células Progenitoras Endoteliais/efeitos dos fármacos , Quercetina/análogos & derivados , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Extremidades/irrigação sanguínea , Flavonoides/farmacologia , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Isquemia/patologia , Lipoproteínas LDL/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quercetina/administração & dosagem , Quercetina/farmacologiaRESUMO
BACKGROUND/AIMS: Dysfunction of endothelial progenitor cell (EPCs) contributes to diabetic vascular disease. We reported that downregulated miR-126 in diabetic patients causes EPC dysfunction. The study was designed to investigate how high glucose (HG) and advanced glycation end products (AGEs) regulate miR-126 expression and whether miR-126 mediates the effects of HG and AGEs on EPCs. METHODS: We first tested the effects of glucose (5.5-50 mM) and AGEs at 50-200 mg/l on EPC proliferation and selected HG at 50 mM and AGEs at 50 mg/l for further experiments. EPCs were stimulated with HG and AGEs, and miR-126 expression was measured by real-time PCR. Reactive oxygen species (ROS) were measured by immunofluorescence microscopy and flow cytometry. IL-6 and TNF-α levels in EPC supernatants were determined by ELISA. The effects of miR-126 on ROS and inflammatory markers under stimulation of HG and AGEs were also assessed. Finally, the effects of inhibitors of PI3K and Akt on AGE-mediated miR-126 expression were examined. RESULTS: HG and AGEs increased IL-6, TNF-α and ROS and decreased miR-126 expression in EPCs. miR-126 negatively regulated IL-6, TNF-α and ROS. miR-126 overexpression reduced and miR-126 inhibition further increased the inflammatory markers and ROS induced by HG and AGEs. Inhibitors of PI3K and Akt further decreased miR-126 expression in the presence of AGEs. CONCLUSIONS: In conclusion, hyperglycemia and AGEs decrease miR-126 expression in EPCs. Recovering miR-126 expression may protect EPCs against dysfunction induced by HG and AGEs.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Progenitoras Endoteliais/efeitos dos fármacos , Glucose/toxicidade , Produtos Finais de Glicação Avançada/toxicidade , MicroRNAs/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Relação Dose-Resposta a Droga , Regulação para Baixo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Retroalimentação Fisiológica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , MicroRNAs/genética , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pancreatic cancer is an aggressive malignancy. The median survival rate remains low, indicating that the identification of novel biomarkers and therapeutic targets is critical. Here, we examined the role of microRNA-182 (miR-182) in pancreatic cancer development. Analysis of human pancreatic cancer specimens and cell lines showed that miR-182 is overexpressed in pancreatic cancer and promotes tumor proliferation and invasion. ß-TrCP2 was confirmed as a direct target of miR-182. Silencing of ß-TrCP2 increased the levels of ß-catenin, which is similar to miR-182 overexpression. Ectopic expression of ß-TrCP2 inhibited the miR-182-induced activation of ß-catenin signaling. The oncogenic effect of miR-182 and its reversal by ß-TrCP2 were confirmed in vivo This study suggests that ß-TrCP and miR-182 may be possible biomarkers and targets for early detection and treatment of pancreatic cancer.