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Cas9-mediated in vivo miRNA silencing

ABSTRACT: Although the advent of Cas9 technology has expanded our ability to precisely edit the genome, manipulating micro RNAs in vivo has been shown to be particularly challenging, especially in the brain. Here, we sought to generate novel tools aiming at targeting and efficiently down regulating defined microRNAs species in a cell-specific manner so that their function in discrete neuronal networks could be investigated. Focusing on miR-124, a micro RNA highly expressed in the mammalian brain and transcribed from three independent chromosomal loci, we designed and validated different gRNAs directed against this miRNA. In vitro, our Cas9 designs show not only a significant reduction in miR-124 levels but also a functional effect on miR-124 silencing. Similarly, when packed into AAV vectors and injected into the mouse cortex, miR-124-Cas9 vectors strongly down regulate miR-124 levels without affecting the expression of other miRNAs. In parallel, levels of endogenous miR-124 targets exhibit a significant increase supporting the release of its silencing activity. To functionally validate our tools, we provide evidences that deletion of miR-124 in the sub ventricular zone altered migration of newly generated neurons into the olfactory bulb. Finally, we also showed that our vectors also modified the Ca2+ permeability of AMPA receptors, a robust functional output downstream of miR-124. These tools are expected to help elucidating miRNA function in complex experimental settings such as brain networks in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE291957 | GEO | 2025/11/24

REPOSITORIES: GEO

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Project description:Increasing evidences support a potential role for the Signal Transducer and Activator of Transcription-3 (STAT3) as a tumor driver in cutaneous T-cell lymphoma (CTCL). However, the mechanisms leading to STAT3 pathway activation in CTCL and how STAT3 activation contributes to lymphomagenesis remains primarily not enough explored. Recently, we found that miR-124, a known STAT3 regulator in cancer, is robustly silenced in Mycosis Fungoides (MF) tumor-stage and CTCL cell lines and we have herein studied whether deregulation of miR-124 contributes to activate STAT3 pathway in CTCL. Material and Methods: DNA methylation status of miR-124 and its expression levels in response to the DNA-demethylating agent azacitidine were evaluated in CTCL cell lines by pyrosequencing analysis. CTCL cell lines were transient transfected with a lentiviral vector encoding miR-124 and transfected cells were analyzed for phosphorylated STAT3 (P-STAT3) levels. The impact on STAT3 signaling was evaluated using expression microarray on CTCL cell lines in both conditions, miR-124 absent (malignant condition) versus miR-124 expressed (lentiviral transduction). Results: A significant promoter methylation and silencing of miR-124 in MF tumor samples and CTCL cell lines was detected. DNA methylation levels of miR-124 in CTCL cell lines were restored (and subsequently miR-124 were overexpressed) after DNA demethylation. Ectopic lentiviral expression of miR-124 downregulated STAT3 in the different CTCL cell lines. Genes differentially expressed regarding miR-124 regulation in CTCL cell lines indicated impact on known cell survival and function pathways and, particularly, intervention on miR-124 expression modulated different STAT3 pathway components. Conclusions: Our study deciphers a novel epigenetic mechanisms regulating STAT3 pathway in CTCL. This might contribute to a better understanding the molecular basis in CTCL development. Deregulation of STAT3 seems to have a major impact on cell survival in CTCL cell lines indicating the potential interest of targeting STAT3 with specific therapies.

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Cas9-mediated in vivo miRNA silencing

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