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Retinoblastoma (RB) is an intraocular tumor in children. Accumulated evidence confirms that microRNAs (miRNAs) exert critical functions in RB. This research aimed to investigate the miR-452-5p function in RB. MiR-452-5p expressions in RB were tested with quantitative real-time polymerase chain reaction (PCR). MiR-452-5p functions in RB were evaluated via Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine assay, flow cytometry, Western blot, and Transwell. MiR-452-5p mechanism in RB was assessed using bioinformatics software Starbase and dual-luciferase reporter gene assay. Meanwhile, miR-452-5p function in RB in vivo was examined by constructing tumor xenografts in nude mice, immunohistochemistry, and Western blot assays. MiR-452-5p was overexpressed in RB tissues and cells, and miR-452-5p expression was positively correlated with RB clinicopathology including the Largest tumor base (mm) and Differentiation. Functionally, miR-452-5p knockdown restrained RB cell proliferation, invasion, epithelial-mesenchymal transition (EMT), and facilitated cell apoptosis. Mechanistically, suppressors of cytokine signaling (SOCS3) knockdown restored the inhibitory effects of miR-452-5p knockdown on RB cells. Meanwhile, in vivo studies further corroborated that miR-452-5p knockdown reduced RB tumor growth, EMT, and accelerated apoptosis in vivo. Also, miR-452-5p knockdown increased SOCS3 protein levels, and decreased phosphorylated Janus kinase 2/Janus kinase 2 (JAK2), phosphorylated signal transducer and activator of transcription 3/signal transducer and activator of transcription 3 (STAT3) in vivo. MiR-452-5p accelerated RB cell growth and invasion by SOCS3/JAK2/STAT3.
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MicroRNAs , Neoplasias da Retina , Retinoblastoma , Animais , Camundongos , Criança , Humanos , Retinoblastoma/genética , Retinoblastoma/patologia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Camundongos Nus , Transdução de Sinais , MicroRNAs/metabolismo , Proliferação de Células , Apoptose , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismoRESUMO
OBJECTIVE: This study was performed to evaluate the outcomes of 25-gauge (25-G) pars plana vitrectomy (PPV) with air tamponade for primary rhegmatogenous retinal detachment (RRD). METHODS: This retrospective consecutive case series included 126 eyes of 125 patients with primary RRD who underwent 25-G PPV with air tamponade. The patients were followed up for at least 6 months following surgery. The main outcome measures were the primary and final anatomical success rates and postoperative complications. RESULTS: The mean age of the 125 patients (80 men and 45 women) was 53.7 ± 10.0 years. The mean follow-up period was 8.3 ± 2.2 months (range, 6-18 months). Twenty-four eyes (19.0%) presented with high myopia, and 13 eyes (10.3%) were pseudophakic. Of the 126 eyes, 37 (29.4%) had inferior breaks, 2 (1.6%) had choroidal detachment, and 86 (68.3%) had macular detachment. The single- and final-operation success rates were 96.0% and 100%, respectively. Postoperative complications included macular hole formation in two eyes. During follow-up, secondary cataract surgery was performed in 27 (23.9%) of the 113 phakic eyes. CONCLUSION: 25-G PPV with air tamponade is effective and safe in treating selected patients with primary RRD with a high anatomical success rate.
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Descolamento Retiniano , Perfurações Retinianas , Masculino , Humanos , Feminino , Adulto , Pessoa de Meia-Idade , Descolamento Retiniano/cirurgia , Descolamento Retiniano/complicações , Estudos Retrospectivos , Acuidade Visual , Vitrectomia/efeitos adversos , Perfurações Retinianas/cirurgia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Resultado do TratamentoRESUMO
To evaluate the outcomes of 25-guage (G) pars plana vitrectomy (PPV) with air tamponade for rhegmatogenous retinal detachment (RRD) with inferior breaks. This retrospective consecutive case series included fifty-two eyes of fifty-two RRD patients with inferior breaks who underwent 25-G PPV with air tamponade. These patients were followed up for at least 6 months following surgery. Primary and final anatomical success rates and postoperative complications were the main outcome measures. The mean age of the patients (39 men and 13 women) was 51.8 ± 11.8 years. There were 49 primary RRDs (94.2%) and three recurrent RRDs (5.8%). The mean follow-up period was 8.2 ± 1.6 months (range: 6-13 months). Sixteen eyes (30.8%) presented with high myopia, and six eyes (11.5%) were pseudophakic. Proliferative vitreous retinopathy grade was C1 in four eyes (7.7%). Of the 52 eyes, two (3.8%) were complicated with choroidal detachment, and forty (76.9%) had the macula detached. The single- and final-operation success rates were 96.2% and 100%, respectively. During follow-up, secondary cataract surgery was performed in eight eyes (17.4%) of the 46 phakic eyes. 25-G PPV with air tamponade is effective in treating selected RRD patients with inferior breaks. Patients can benefit from early visual recovery and less complications.
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PURPOSE: This study aimed to investigate the association of Demodex infestation with pediatric chalazia. METHODS: In a prospective study, 446 children with chalazia and 50 children with non-inflammatory eye disease (controls) who underwent surgical treatment were enrolled from December 2018 to December 2019. Patient ages ranged from 7 months to 13 years old. All patients underwent eyelash sampling for light microscope examination, and statistical correlation analysis between Demodex infestation and chalazia, including the occurrence, recurrence, and course of disease, morphological characteristics, and meibomian gland dysfunction (MGD) in chalazia patients was performed. RESULTS: Demodex was found in 236 (52.91%) patients with chalazia and zero control patients. Demodicosis was significantly more prevalent in chalazia patients than the control group (P < 1 × 10- 14). Recurrent chalazia (P = 0.006) and skin surface involvement (P = 0.029) were highly correlated with Demodex infestation. Demodicosis was also associated with multiple chalazia (P = .023) and MGD(P = .024). However, Demodex infestation was comparable in the course of disease (P = 0.15), seasonal change (P = 0.68) and blepharitis subgroups (P = 0.15). Within the group of chalazia patients who underwent surgical removal of cysts, 4 (0.9%) patients with concurrent demodicosis experienced recurrence. CONCLUSIONS: Demodex infestation was more prevalent in pediatric chalazia patients than healthy children, and was associated with recurrent and multiple chalazia. Demodicosis should be considered as a risk factor of chalazia. In children with chalazia, Demodex examination and comprehensive treatment of Demodex mites should be applied to potentially prevent recurrence.
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Calázio , Infecções Oculares Parasitárias , Infestações por Ácaros , Ácaros , Animais , Calázio/complicações , Calázio/diagnóstico , Calázio/epidemiologia , Criança , Infecções Oculares Parasitárias/diagnóstico , Infecções Oculares Parasitárias/epidemiologia , Infecções Oculares Parasitárias/cirurgia , Humanos , Lactente , Infestações por Ácaros/complicações , Infestações por Ácaros/epidemiologia , Estudos ProspectivosRESUMO
Recurrent epithelial erosion and refractory corneal ulcer are the clinical features of diabetic keratopathy (DK), which eventually lead to corneal scar and visual disturbance. In this study, we sought to determine the abnormalities of cell junction in diabetic corneal epithelial cells and the effect of high glucose on the ß-catenin/E-cadherin complex. Corneal histology showed that corneal epithelial cells of high glucose mice were loosely arranged, and the immunohistochemistry showed that the expression of E-cadherin decreased, the levels of ß-catenin increased in nuclear. High glucose-induced degradation and endocytosis of E-cadherin of corneal epithelial cells reduce the formation of ß-catenin/E-cadherin complex and promote the nuclear translocation of ß-catenin. Moreover, high glucose also activated the transcription and expression of matrix metallopeptidase and snail, which interfered with the adhesion of corneal epithelial cells to the basement membrane. These findings reveal that DK is associated with the dissociation of cell junctions. The maintenance of the stability of the ß-catenin/E-cadherin complex may be a potential therapeutic target of refractory corneal ulcers in patients with diabetes.
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Caderinas/metabolismo , Núcleo Celular/metabolismo , Córnea/metabolismo , Córnea/patologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Endocitose , beta Catenina/metabolismo , Animais , Membrana Basal/metabolismo , Glicemia/metabolismo , Peso Corporal , Diferenciação Celular , Células Epiteliais/patologia , Epitélio Corneano/patologia , Comportamento Alimentar , Metaloproteinase 10 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Proteólise , Fatores de Transcrição da Família Snail/metabolismo , CicatrizaçãoRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system, accounting for 48.6% of malignant tumors. The current standard treatment plan includes the widest range of safe surgical resection, supplemented by local brain radiotherapy and temozolomide concurrent chemotherapy; this can cause serious side effects. Even so, the median survival time of GBM patients is only 8 months, and the 5-year survival rate is only 5.5%. It is imminent to find new treatments. Early studies have shown that chicken and zebrafish embryos can reprogram cancer cells into a non-tumorigenic phenotype through the embryonic microenvironment. However, the effect of embryonic stem cell microenvironment on GBM and its possible mechanism are not clear. METHODS: In this study, the glioblastoma cell line, U118, in the brain was investigated. There were four experimental groups: GB, GE, GA and GT. U118 cells were harvested after culturing for 72 hours. Cell proliferation, apoptosis, reactive oxygen species (ROS) were examined using vasculogenic mimicry assays, quantitative real-time polymerase chain reaction (QRT-PCR), western blotting (WB) and flow cytometry. The differences in the biological function of U118 cells and the PI3K/protein kinase B (AKT) signaling pathway were compared between the groups. RESULTS: Compared with the GB control group, the GE co-culture group and GT chemotherapy group showed reduced cell proliferation, increased apoptosis, increased ROS, as well as decreased or inhibited vasculogenic mimicry. Expressions of cyclin B1 and cyclin D1 were also notably reduced, while that of Bax, Bcl-2, p53, Caspase-3, GSK-3ß, p21, and p27 were significantly increased. Moreover, the expression of PI3K, AKT, and mTOR were markedly decreased, whereas expression of PTEN increased considerably. Also, the expression of positive regulatory factors significantly increased, however negative regulatory factors decreased in the GA group compared to the GE group. CONCLUSIONS: The ESC microenvironment reverses glioma malignancy, partially via inhibition of the PI3K signaling pathway. Our study may have a significant impact and important clinical implications for cell therapy in the treatment of glioma.
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OBJECTIVE: To investigate whether DNMT1 protein induces retinoblastoma proliferation by silencing MEG3 gene. METHODS: Two retinoblastoma cell lines (HXO-RB44 and SO-RB50) and a normal human retinal pigment epithelial (RPE) cell line were transfected with the plasmid pcDNA-DNMT1 or si-DNMT1 for up-regulating or interference of DNMT1 expression, and with pcDNA-MEG3 or si-MEG3 for up-regulating or interference of MEG3 expression. Western blotting was used to detect the changes in the expression of DNMT1 protein in the transfected cells, and CCK-8 and EdU assays were used to detect the changes in cell proliferation. Real-time quantitative PCR (qRT-PCR) was performed to detect MEG3 expression in SO-RB50 and HXO-RB44 cells after transfection, and the methylation level of MEG3 gene promoter after interference of DNMT1 expression was detected using methylation-specific PCR. RESULTS: SO-RB50 and HXO-RB44 cells showed significantly increased expression of DNMT1 protein as compared with normal RPE cells (P < 0.05). In HXO-RB44 cells, transfection with pcDNADNMT1 resulted in significantly increased expression of DNMT1 protein, enhanced cell proliferation ability, and significantly reduced expression of MEG3 (P < 0.05). In SO-RB50 cells, transfection with si-DNMT1 significantly reduced the expression of DNMT1 protein, suppressed the cell proliferation, and increased MEG3 expression (P < 0.05). Interference of DNMT1 significantly reduced the methylation level of MEG3 gene promoter. After reversing the regulatory effect of DNMT1 on MEG3 gene, DNMT1 protein showed significantly weakened ability to regulate retinoblastoma cell proliferation (P < 0.05). CONCLUSIONS: In retinoblastoma cells, the up-regulation of DNMT1 protein induces promoter methylation and inactivation of MEG3 gene and eventually leads to abnormal cell proliferation.
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Neoplasias da Retina , Retinoblastoma , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , DNA (Citosina-5-)-Metiltransferase 1 , Humanos , RNA Longo não Codificante/genética , Neoplasias da Retina/genética , Retinoblastoma/genéticaRESUMO
The vitreous substitute for proliferative vitreoretinopathy (PVR) surgery remains an unmet clinical need in ophthalmology. In our study, we developed an in situ formed hydrogel by crosslinking polyvinyl alcohol (PVA) and chitosan as a potential vitreous substitute. 5-fluorouracil (5-FU) Poly (lactic-co-glycolic acid) (PLGA) microspheres were developed and loaded onto the PVA/chitosan hydrogels to treat PVR. In vitro, PVA/chitosan hydrogels at four concentrations were subjected to morphological, physical, rheological analyses, and cytotoxicity was evaluated together with the characterization of 5-FU PLGA microspheres. In vivo, pharmacologically induce PVR rabbits were performed a vitrectomy. In the PVA group, 3% PVA/chitosan hydrogel was injected into the vitreous cavity. In the PVA/MS group, 3% PVA/chitosan hydrogel and 5-FU PLGA microspheres were injected. In the Control group, phosphate-buffered saline was injected. Therapeutic efficacy was evaluated with postoperative examinations and histological analyses. This study demonstrated that the 3% PVA/chitosan hydrogel showed properties similar to those of the human vitreous and could be a novel in situ crosslinked vitreous substitute for PVR. Loading 5-FU PLGA microspheres onto this hydrogel may represent an effective strategy to improve the prognosis of PVR.
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Sistemas de Liberação de Medicamentos , Fluoruracila , Hidrogéis , Microesferas , Vitreorretinopatia Proliferativa , Animais , Linhagem Celular , Fluoruracila/química , Fluoruracila/farmacologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Coelhos , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologiaRESUMO
PURPOSE: To compare tear protein markers between normal subjects and patients with dry eye (DE) and high and low lymphotoxin-alpha (LT-α) levels. DESIGN: Prospective cross-sectional study. METHODS: Patients with DE were divided into low (≤700 pg/mL) and high (>700 pg/mL) LT-α groups. Twelve protein markers were measured by microsphere-based immunoassay and ocular surface parameters were determined in right eyes (33 high LT-α DE, 27 low LT-α DE, and 20 control eyes) and left eyes (21 high LT-α DE, 39 low LT-α DE, and 20 control eyes). RESULTS: In both eyes, tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-1ß, IL-1 receptor antagonist (IL-1Ra), IL-17A, and IL-12/23 p40 levels in high LT-α DE were significantly higher (P < .01) than in low LT-α DE. Significant correlations identified in high LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = 0.43, P = .013), IL-1ß (R = 0.48, P = .005), and IL-12/23 p40 (R = 0.50, P = .003), IL-12/23 p40 with ocular surface disease index (R = 0.35, P = .049), and epidermal growth factor with corneal fluorescein staining score (R = -0.36, P = .038). Significant correlations in low LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = -0.39, P = .046), TNF-α (R = -0.39, P = .047), and IL-17A (R = -0.48, P = .013), ocular surface disease index with TNF-α (R = -0.47, P = .017) and IL-17A (R = -0.46, P = .018), and IL-6 with tear breakup time (R = -0.40, P = .044). Lastly, IL-1Ra levels significantly increased in DE patients, positively correlated with temporal conjunctival hyperemia index, and negatively correlated with Schirmer I test (P < .05). CONCLUSIONS: Our study identified tear IL-1Ra level as a potential biomarker to replace the Schirmer I test. Multiple tear protein marker levels increased in high LT-α DE, indicating that high LT-α DE might have a different pathogenesis.
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Síndromes do Olho Seco/metabolismo , Linfotoxina-alfa/biossíntese , Lágrimas/metabolismo , Adulto , Biomarcadores/metabolismo , Estudos Transversais , Síndromes do Olho Seco/diagnóstico , Feminino , Humanos , Imunoensaio , Proteína Antagonista do Receptor de Interleucina 1/biossíntese , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
The maturation state of dendritic cell (DC) plays an important role in immune activities. Previously we had found that NF-κB (p65) pathway could promote DC maturation and subsequent immune effects. But the upstream mechanism of this pathway was still unclear. Extracellular adenosine triphosphate (ATP) activating its receptor P2X7R has recently been considered as the fourth signal to activate T lymphocytes. Here we aimed to find out the connection between P2X7R and NF-κB (p65) pathway in DC maturation. Results showed that the expression of P2X7R and the intracellular ATP levels were increased along with the maturation of DC. P2X7R agonist stimulated the morphological changes of DCs into the appearance of mature DCs, and promoted the expression of NF-κB (p65), as well as the release of IFN-γ and IL-12. Whereas, P2X7R inhibitor had the opposite influences. Co-immunoprecipitation assay confirmed the binding of P2X7R and NF-κB (p65). Our study suggested that extracellular ATP could promote DC maturation and release of inflammatory cytokines through the binding of P2X7R and NF-κB (p65). This is the first study to show the P2X7R-NF-κB (p65) pathway in DC. Interference with this pathway may be able to regulate immune responses in areas like infectious diseases, inflammation, transplantation, tumor and autoimmune diseases. In addition, intracellular ATP level could be a new indicator of the maturation state of DC.
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Trifosfato de Adenosina/metabolismo , Medula Óssea/metabolismo , Células Dendríticas/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/metabolismo , Animais , Diferenciação Celular/fisiologia , Citocinas/metabolismo , Feminino , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Linfócitos T/metabolismoRESUMO
Dry eye disease (DED), a multifactorial ocular surface disorder affecting millions of individuals worldwide, is characterized by inflammation and damage to the ocular surface. It is unclear whether corneal autophagy participates in ocular surface inflammation observed in DED. To test this involvement, dry eye (DE) was induced in female C57BL/6 mice housed in a controlled environment by subcutaneous injection of scopolamine. Expression of the autophagy-related proteins LC3B and ATG5 and activation of autophagy were detected in the corneas of these mice. Treatment with LYN-1604, an activator of autophagy, alleviated the clinical indications in DE mice, including tear production and corneal fluorescence staining. LYN-1604 also reduced the corneal levels of inflammatory response products, including tumor necrosis factor alpha (TNF-α) and matrix metalloproteinases-3 and -9. By contrast, treatment of DE mice with the autophagy inhibitor 3-MA, exacerbated the clinical indications of DE and increased the levels of inflammatory response products. This is the first study to show that autophagy could regulate the level of ocular surface inflammation, suggesting that agents that regulate autophagy could relieve ocular surface inflammation and treat DED.
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Autofagia/fisiologia , Córnea/fisiologia , Síndromes do Olho Seco/fisiopatologia , Ceratite/fisiopatologia , Animais , Proteína 5 Relacionada à Autofagia/metabolismo , Biomarcadores/metabolismo , Western Blotting , Síndromes do Olho Seco/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Ceratite/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Lágrimas/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The aim of the present study was to investigate the role of histatin 1 (Hst1) in human corneal epithelial cells (HCECs) exposed to ultraviolet (UV) radiation. Prior to UV irradiation for various durations, HCECs were pre-treated with different concentrations of Hst1 and the effect on cell apoptosis and cell viability were examined by flow cytometry, alamarBlue® and MTT assays to determine the optimal concentration of Hst1 and UV dose. Cells were then subjected to quantitative PCR, ELISA and western blot analysis to determine the expression of cell damage-associated genes. HCECs exposed to UV light for 1 h displayed decreased viability when compared to that of control cells, and a 3 h UV exposure markedly increased the apoptotic rate of HECEs, while apoptosis was inhibited by pre-treatment with Hst1. UV radiation downregulated expression of insulin-like growth factor (IGF)-1 and B-cell lymphoma 2 (Bcl-2), while it upregulated Bcl-2-associated X protein (Bax) expression. Hst1 protected HCECs against UV-induced damage by upregulating the expression of IGF-1 protein and increasing the Bcl-2/Bax ratio. In conclusion, Hst1 may prevent UV-induced damage to corneal epithelial tissue injury and promote its healing.
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Classically activated macrophages (M1) are proinflammatory effectors and closely related to the progression of neurotoxicity. As a powerful psychostimulant and addictive drug, methamphetamine (Meth) abuse could result in long-lasting abnormalities in retina. This study investigated the effect of Meth at nontoxic concentration on macrophage activation state and its resultant toxicity to photoreceptor cells. Results showed that cytotoxicity was caused by Meth on 661 W cells after coculturing with RAW264.7 macrophage. RAW264.7 cells tended to switch to the M1 phenotype, releasing more proinflammatory cytokines after treatment with Meth. Meth could also upregulate the M1-related gene and protein expression. Our study demonstrated that Meth promoted macrophage polarization from M0 to M1 and induced inflammatory response, providing the scientific rationale for the photoreceptor cell damage caused by the Meth abuse.
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Estimulantes do Sistema Nervoso Central/toxicidade , Macrófagos/efeitos dos fármacos , Metanfetamina/toxicidade , Células Fotorreceptoras/efeitos dos fármacos , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Fragmentação do DNA , L-Lactato Desidrogenase/metabolismo , Macrófagos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Células Fotorreceptoras/metabolismoRESUMO
Previous studies have shown that microRNA-186 (miR-186) is overexpressed in various human cancers and is associated with the regulation of the carcinogenic processes. However, the underlying mechanisms of this microRNA in melanoma remain largely unknown. In the present study, the overexpression of miR-186 was identified in melanoma tissues and melanoma cells compared to the expression of miR-186 in the matched tumor adjacent tissues and normal human epidermal melanocytes. Overexpression of miR-186 promoted the proliferation and anchorage-independent growth of melanoma cells, whereas inhibition of miR-186 reduced this effect. Bioinformatics analysis also revealed cylindromatosis (CYLD), a putative tumor suppressor, to be a potential target of miR-186. Luciferase reporter assays showed that miR-186 directly targeted the 3'-untranslated regions of CYLD messenger RNA. Additional experiments showed that overexpression of miR-186 promoted the proliferation of melanoma cells, which was consistent with the inhibitory effects induced by knockdown of CYLD. In summary, the present study indicated that miRNA-186 plays a crucial role in melanoma growth and its oncogenic effect is mediated chiefly through the direct suppression of CYLD expression.
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Properly controlled corneal epithelial wound healing is critical for health of cornea, which involves cell proliferation, migration, anchoring and differentiation. Sodium hyaluronate (SH) has been proven to exert beneficial pharmacological effect on corneal wound healing, though the underlying mechanism remained open to investigation. MicroRNAs (miRNAs) are small single-stranded RNAs that could bind to 3'UTR of mRNAs of target genes. The multi-target regulation of miRNAs may favor treatment of corneal wound given the complicated processes implicated in the healing process, which has inspired initiatives to develop miRNA therapy in corneal wound healing. In this light, we used miRNAs profiling to detect whether miRNAs are also implicated in the mechanism underlying the stimulatory effect of SH on corneal epithelial wound healing. We found miR-18a was most susceptible to SH treatment, the target prediction of which were enriched in a bunch of pathways implicated in corneal wound healing. Connective tissue growth factor (CTGF) was found to be overrepresented in most significant enriched pathways and was experimentally confirmed as a bona fide target of miR-18a, which modulated cell migration and proliferation of human corneal epithelial cells.
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Movimento Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Epitélio Corneano/citologia , Ácido Hialurônico/farmacologia , MicroRNAs/genética , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Antagomirs/farmacologia , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
MicroRNAs (miRNAs) are short, non-coding RNAs with post-transcriptional regulatory function, playing crucial roles in cancer development and progression of human melanoma. Previous studies have indicated that miR-769 was implicated in diverse biological processes. However, the underlying mechanism of miR-769 in human melanoma has not been intensively investigated. In this present study, we aimed to investigate the role of miR-769 and its target genes in human melanoma. We found that miR-769 expression was strongly increased in human melanoma cells and clinical tissues compared with their corresponding controls. Overexpression of miR-769 promoted cell proliferation in human melanoma cell line A375, whereas miR-769-in reverses the function. Glycogen synthase kinase-3 Beta (GSK3B), a potential target gene of miR-769, and was validated by luciferase assay. Further studies revealed that miR-769 regulated cell proliferation of human melanoma by directly suppressing GSK3B expression and the knockdown of GSK3B expression reversed the effect of miR-769-in on human melanoma cell proliferation. In summary, our data demonstrated that miR-769 might act as a tumor promoter by targeting GSK3B during development of human melanoma.
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Glicogênio Sintase Quinase 3 beta/metabolismo , Melanoma/genética , Melanoma/patologia , MicroRNAs/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Regiões 3' não Traduzidas/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/enzimologia , MicroRNAs/genética , Ligação Proteica , Neoplasias Cutâneas/enzimologia , Regulação para Cima/genéticaRESUMO
RelA, the most important regulator of NF-kB activity, and its mechanisms in keratoplasty immune rejection have not been fully investigated. In the present study, lentivirus-mediated silencing of RelA expression in a bone marrow-derived dendritic cell (BMDC) model was tested. The BMDCs were transfected with RelA-shRNA to induce an immature, maturation-resistant and tolerogenic phenotype, while not significantly changing IFN-γ, IL-10 and IL-17 expression. A fully allogeneic rat cornea transplant model was established for in vivo studies. The allograft mean survival time (MST) of lv-shRelA-DC injection groups were significantly longer than the untreated BMDC group and control group. The corneal opacity and neovascularization scale of the lv-shRelA-DC injection groups were slight compared to pair control others. Postoperative flow cytometric analysis revealed that the percentage of Treg positive cells was dramatically increased in animals that received an lv-shRelA-DC injection. ELISA and qRT-PCR analyses of serum showed that IFN-γ and IL-17 expression were suppressed by lv-shRelA-DC treatement. In vivo experiments demonstrated that IL-10 induced immunosuppression was partly attributed to injection of lv-shRelA-DC throughout the experiment, differing from the general anti-inflammatory factors. Luciferase and Chromatin IP evaluation showed that RelA knockdown in BMDCs significantly reduces DNA binding to IFN-γ, IL-10 and the IL-17 promoter and inhibited of transcriptional activity. Taken together, this study illustrates a significant role of RelA in mediating the corneal neovascularization by affecting IL-17 expression. Our comprehensive analysis shows that the significant role of RelA provides a novel and feasible therapeutic approach for the prevention of corneal allograft rejection.
Assuntos
Neovascularização da Córnea/imunologia , Transplante de Córnea , Tolerância Imunológica/imunologia , Fator de Transcrição RelA/imunologia , Imunologia de Transplantes/imunologia , Aloenxertos , Animais , Imunoprecipitação da Cromatina , Células Dendríticas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Imuno-Histoquímica , Interleucina-10/imunologia , Interleucina-17/imunologia , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Fator de Transcrição RelA/metabolismoRESUMO
The aberrant expression of MEG3 has been found in some types of cancers; however, little is known concerning the function of MEG3 in retinoblastoma. To elucidate the roles of MEG3 in retinoblastoma, MEG3 expression was quantified in 63 retinoblastoma samples and corresponding nontumor tissues in this work. Moreover, retinoblastoma cell lines were transfected with pcDNA3.1-MEG3 or si-MEG3, after which proliferation, apoptosis, and expression of ß-catenin were assayed. TOP-Flash reporter assay was also used to investigate the activity of the Wnt/ß-catenin pathway. The results showed that MEG3 was downregulated in retinoblastoma tissues, and the level of MEG3 was negatively associated with IIRC stages and nodal or distant metastasis. More importantly, Kaplan-Meier survival analysis demonstrated that patients with low MEG3 expression had poorer survival and multivariate Cox regression analysis revealed that MEG3 was an independent prognostic factor in retinoblastoma patients. We also observed that MEG3 expression can be modulated by DNA methylation by using 5-aza-CdR treatment. In addition, overexpression of MEG3 suppressed proliferation, promoted apoptosis, and influences the activity of the Wnt/ß-catenin pathway in retinoblastoma cell lines. Furthermore, we found that Wnt/ß-catenin pathway activator rescued the anticancer effect of MEG3 in retinoblastoma. In conclusion, our study for the first time demonstrated that MEG3 was a tumor suppressor by negatively regulating the activity of the Wnt/ß-catenin pathway in the progression of retinoblastoma and might serve as a prognostic biomarker and molecular therapeutic target.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , RNA Longo não Codificante/genética , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Via de Sinalização Wnt/genética , Apoptose/genética , Área Sob a Curva , Biomarcadores Tumorais/genética , Western Blotting , Proliferação de Células/genética , Criança , Pré-Escolar , Progressão da Doença , Regulação para Baixo , Feminino , Genes Supressores de Tumor/fisiologia , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Reação em Cadeia da Polimerase , Modelos de Riscos Proporcionais , Curva ROC , Neoplasias da Retina/genética , Neoplasias da Retina/mortalidade , Retinoblastoma/genética , Retinoblastoma/mortalidadeRESUMO
BACKGROUND: This prospective study investigated the safety and efficacy of a therapeutic method of treating pterygium complicated with conjunctivochalasis, using pterygium excision and conjunctival autotransplantation combined with sclera fixation, followed by therapeutic contact lens application. METHODS: Fifty-seven patients (83 eyes) diagnosed as pterygium complicated with conjunctivochalasis, at our hospital from July 2011 to June 2012, were selected. Patients were treated with pterygium excision and conjunctival autotransplantation combined with sclera fixation surgery, then therapeutic bandage contact lenses were applied. The efficacy of simultaneous surgery was evaluated based on vision changes, tear dynamics, and other complications. Histopathological changes were investigated on removed bulbar conjunctival tissue, using hematoxylin eosin (HE) and Masson's trichrome staining. RESULTS: (1) Three months after the operation, the success of simultaneous surgery in the treatment of pterygium was 97.6 %, and the recurrence was 2.4 %. Based on subjective evaluation, the success of the simultaneous treatment of conjunctivochalasis was 95.2 %, and failure was 4.8 %. Based on objective evaluation, the success rate was 94.0 % and the recurrence rate was 6.0 %. (2) Visual acuity of the 83 eyes was significantly improved after surgery, and was statistically significant (X 2 = 10.29, P < 0.05). (3) Three months after surgery, the height and integrity of the tear meniscus, tear film break-up time, and chloramphenicol test results of the 83 eyes were significantly improved and there was a statistically significant difference (X 2 the height and integrity of tear meniscus = 147.24, X 2 tear film break-up time = 81.17, X 2 chloramphenicol test = 17.41, P < 0.01). (4) Complications after the operation such as granulation hyperplasia, constrictive fornix, oculomotor defect, and other complications were not observed. (5) Pathological observations, using HE and Masson's trichrome staining of removed bulbar conjunctival tissue, showed several pathological changes, including obvious squamous epithelial hyperplasia, parakeratosis, basal cell pigmentation, lamina propria hemorrhage, infiltration of lymphocytes, and reduction of elastic fibers and collagen fibers. CONCLUSION: Pterygium excision and conjunctival autotransplantation, combined with sclera fixation followed by therapeutic contact lens use was safe, effective, and suitable for simultaneous treatment of pterygium complicated with conjunctivochalasis.