RESUMO
Early treatment of retinoblastoma (RB) has significantly improved clinical outcomes. N6-methyladenosine (m6A) methylation is crucial for cancer progression. Thus, we investigated the role of FTO-dependent demethylation in RB and its underlying mechanisms. The biological behavior of RB cells was analyzed using cell counting kit-8, colony formation analysis, transwell assay, flow cytometry, and western blot analysis. m6A modification was evaluated using methylated RNA immunoprecipitation and dual-luciferase reporter assays, and E2F3 stability was assessed using Actinomycin D. The roles of FTO and E2F3 were also elucidated in vivo. These results indicated that FTO was highly expressed in RB cells with low m6A levels. FTO knockdown inhibited RB cell growth, migration, invasion, and epithelial-mesenchymal transition and arrested the cell cycle at the G0/G1 phase. Mechanistically, FTO interference promoted m6A methylation of E2F3, which was recognized by YTHDF2, thereby reducing mRNA stability. E2F3 overexpression partially rescued the effects of FTO knockdown on biological behavior. Moreover, FTO knockdown reduced tumor weight, tumor volume, ki67 expression, and tumor cell infiltration by mediating E2F3. Taken together, FTO silencing inhibited the malignant processes of RB by suppressing E2F3 in an m6A-YTHD2-dependent manner. These findings suggest that FTO is a novel therapeutic target for RB.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Fator de Transcrição E2F3 , Neoplasias da Retina , Retinoblastoma , Humanos , Adenosina , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Ciclo Celular , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F3/metabolismo , Retinoblastoma/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Epithelial-melancholy transition (EMT) is the main cause of organ fibrosis and a common pathogenetic mechanism in most cataracts. This study aimed to explore the molecular mechanism of Toll-like receptor (TLR)-3 in the occurrence and development of post-cataract EMT and to provide new ideas for the prevention and treatment of posterior capsule opacification (PCO). In the presence or absence of TLR3, the human lens epithelial cell (LEC) line, SRA01/04, was treated with the transforming growth factor (TGF)-ß2. Cell counting kit-8 (CCK-8) and Transwell assays were used to analyze the cell proliferation, migration, and invasion. The expression levels of proteins and RNAs were detected by western blotting and quantitative polymerase chain reaction (qPCR) experiments. Functional gain and loss studies showed that TLR3 regulates the proliferation, migration, and invasion of LECs and EMT induced by TGF-ß2. Moreover, TLR3 regulates the expression of Jagged-1, Notch-1, and Notch-3 These findings indicate that TLR3 prevents the progression of lens fibrosis by targeting the Jagged-1/Notch signaling pathway to regulate the proliferation, migration, and invasion of LECs, and TGF-ß2-induced EMT. Therefore, the TLR3-Jagged-1/Notch signaling axis may be a potential therapeutic target for the treatment of fibrotic cataracts.
Assuntos
Opacificação da Cápsula , Receptores Notch , Receptor 3 Toll-Like , Opacificação da Cápsula/genética , Opacificação da Cápsula/metabolismo , Opacificação da Cápsula/patologia , Movimento Celular/genética , Proliferação de Células/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Fibrose , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/genética , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
pH plays an essential role in virtually all biological processes, which thus needs to be tightly regulated. Especially small changes in pH value in biological systems will affect the normal metabolic functions of animals and plants. Therefore, it is very important to accurately track changes in pH in biological systems. Herein, we rationally fabricated a new molecular pH probe (NBD-pbz) based on conjugation of the 2-Piperazin-1-yl-1,3-benzothiazole (pbz) and 7-nitro-1,2,3-benzoxadiazole (NBD) building blocks. The following test on NBD-pbz revealed that it can offer a sensitive and selective tracking of pH changes from 3.2 to 7.6 with pKa 5.51, and not only in eukaryotic cells (especially the imaging of ocular tumor cell OCM-1) and zebrafish but also in mung bean sprouts via fluorometric turn on response and an intramolecular charge transfer (ICT) based working mechanism strategy. Therefore, this NBD-pbz probe not only replenishes the current repertory of molecular pH probes but also extends the application of pH probes to monitor pH variation in the plant kingdom, which will undoubtedly arouse greater research interest in the field.