RESUMO
The roles of specific microRNAs (miRNA) in oligodendrocyte (OL) differentiation have been studied in depth. However, miRNAs in OL precursors and oligodendrocyte progenitor cells (OPCs) have been less extensively investigated. MiR-145-5p is highly expressed in OPCs relative to differentiating OLs, suggesting this miRNA may serve a function specifically in OPCs. Knockdown of miR-145-5p in primary OPCs led to spontaneous differentiation, as evidenced by an increased proportion of MAG+ cells, increased cell ramification, and upregulation of multiple myelin genes including MYRF, TPPP, and MAG, and OL cell cycle exit marker Cdkn1c. Supporting this transition to a differentiating state, proliferation was reduced in miR-145-5p knockdown OPCs. Further, knockdown of miR-145-5p in differentiating OLs showed enhanced differentiation, with increased branching, myelin membrane production, and myelin gene expression. We identified several OL-specific genes targeted by miR-145-5p that exhibited upregulation with miR-145-5p knockdown, including myelin gene regulatory factor (MYRF), that could be regulating the prodifferentiation phenotype in both miR-145 knockdown OPCs and OLs. Indeed, spontaneous differentiation with knockdown of miR-145-5p was fully rescued by concurrent knockdown of MYRF. However, proliferation rate was only partially rescued with MYRF knockdown, and overexpression of miR-145-5p in OPCs increased proliferation rate without affecting expression of already lowly expressed differentiation genes. Taken together, these data suggest that in OPCs miR-145-5p both prevents differentiation at least in part by preventing expression of MYRF and promotes proliferation via as-yet-unidentified mechanisms. These findings clarify the need for differential regulation of miR-145-5p between OPCs and OLs and may have further implications in demyelinating diseases such as multiple sclerosis where miR-145-5p is dysregulated.
Assuntos
Diferenciação Celular/genética , MicroRNAs/genética , Bainha de Mielina/genética , Células Precursoras de Oligodendrócitos/patologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Bainha de Mielina/patologia , Neurogênese/genética , Oligodendroglia/patologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/genéticaRESUMO
Strategies for treating progressive multiple sclerosis (MS) remain limited. Here, we found that miR-145-5p is overabundant uniquely in chronic lesion tissues from secondary progressive MS patients. We induced both acute and chronic demyelination in miR-145 knockout mice to determine its contributions to remyelination failure. Following acute demyelination, no advantage to miR-145 loss could be detected. However, after chronic demyelination, animals with miR-145 loss demonstrated increased remyelination and functional recovery, coincident with altered presence of astrocytes and microglia within the corpus callosum relative to wild-type animals. This improved response in miR-145 knockout animals coincided with a pathological upregulation of miR-145-5p in wild-type animals with chronic cuprizone exposure, paralleling human chronic lesions. Furthermore, miR-145 overexpression specifically in oligodendrocytes (OLs) severely stunted differentiation and negatively impacted survival. RNAseq analysis showed altered transcriptome in these cells with downregulated major pathways involved in myelination. Our data suggest that pathological accumulation of miR-145-5p is a distinctive feature of chronic demyelination and is strongly implicated in the failure of remyelination, possibly due to the inhibition of OL differentiation together with alterations in other glial cells. This is mirrored in chronic MS lesions, and thus miR-145-5p serves as a potential relevant therapeutic target in progressive forms of MS.
Assuntos
Doenças Desmielinizantes , Modelos Animais de Doenças , Camundongos Knockout , MicroRNAs , Remielinização , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Remielinização/genética , Camundongos , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Humanos , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Recuperação de Função Fisiológica , Masculino , Camundongos Endogâmicos C57BL , Cuprizona/toxicidade , Feminino , Doença Crônica , Bainha de Mielina/metabolismoRESUMO
Aim: Cell-free DNA in the plasma is known to be a potential biomarker for noninvasive diagnosis of oncogenic mutations. The authors aimed to design an optimized padlock probe-based hyperbranched rolling circle amplification biosensor to detect the KRAS G12D mutation using fluorescence and colorimetric methods. Methods: Single-factor experiments, Plackett-Burman design and response surface methodology were applied to optimize the padlock probe-based hyperbranched rolling circle amplification reaction. Results: The maximum fluorescence intensity was achieved at a padlock probe concentration of 1.5 pM and target concentration of 9 pM at 38°C ligation temperature. The proposed biosensor has a low detection limit of 60 fM of target DNA and a linear response in the concentration range of 60 fM to 0.2 pM. Conclusion: The results indicated the power of these assays to detect KRAS point mutations in liquid state reactions.
Assuntos
Bioensaio/métodos , Técnicas Biossensoriais , Colorimetria , Corantes Fluorescentes/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Ouro/química , Humanos , Nanopartículas Metálicas/química , Espectrofotometria UltravioletaRESUMO
BACKGROUND: The possibility of using a specific nanoparticle in nanomedicine highly depends on its biodistribution profile and biocompatibility. Due to growing demand for iron oxide nanoparticles (IONPs) and dendrimers in biomedical applications, this study was performed to assess the biodistribution, pharmacokinetics, and toxicity of dendrimer-coated iron oxide nanoparticles (G4@IONPs). MATERIALS AND METHODS: IONPs were synthesized via co-precipitation and coated with the fourth generation (G4) of polyamidoamine (PAMAM) dendrimer. To determine the biodistribution, 5 mg/mL G4@IONPs suspension was intraperitoneally injected into tumor-bearing BALB/c mice, and iron levels in blood and various organs, including the lung, liver, brain, heart, tumor, and kidney, were measured by inductively coupled plasma mass spectrometry (ICP-MS) at 4, 8, 12, and 24 h after injection. Also, to investigate the toxicity of G4@IONPs, different concentrations of G4@IONPs were injected into BALB/c mice, and blood, renal, and hepatic factors were measured. Furthermore, histopathological staining was performed to investigate the effect of G4@IONPs on the liver and kidney tissues. RESULTS: The results showed that the iron content was higher in the kidney, liver, and lung tissues 24 h after injection. Toxicity assessments revealed a significant increase in blood urea nitrogen (BUN) and direct bilirubin at the concentration of 10 mg/kg. Also, in this concentration, histopathological abnormalities were detected in liver tissue. CONCLUSION: Although more systematic studies are still required, our results encouraged the future investigations of G4@IONPs in biomedical applications.