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1.
Proc Natl Acad Sci U S A ; 120(30): e2216658120, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37463203

RESUMO

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.


Assuntos
Epilepsia , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Canais de Sódio Disparados por Voltagem , Humanos , Camundongos , Ratos , Animais , Células-Tronco Pluripotentes Induzidas/metabolismo , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Canais de Sódio Disparados por Voltagem/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.3/genética
2.
Brain Commun ; 6(1): fcad355, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38204971

RESUMO

MicroRNAs have emerged as important regulators of the gene expression landscape in temporal lobe epilepsy. The mechanisms that control microRNA levels and influence target choice remain, however, poorly understood. RNA editing is a post-transcriptional mechanism mediated by the adenosine acting on RNA (ADAR) family of proteins that introduces base modification that diversifies the gene expression landscape. RNA editing has been studied for the mRNA landscape but the extent to which microRNA editing occurs in human temporal lobe epilepsy is unknown. Here, we used small RNA-sequencing data to characterize the identity and extent of microRNA editing in human temporal lobe epilepsy brain samples. This detected low-to-high editing in over 40 of the identified microRNAs. Among microRNA exhibiting the highest editing was miR-376a-3p, which was edited in the seed region and this was predicted to significantly change the target pool. The edited form was expressed at lower levels in human temporal lobe epilepsy samples. We modelled the shift in editing levels of miR-376a-3p in human-induced pluripotent stem cell-derived neurons. Reducing levels of the edited form of miR-376a-3p using antisense oligonucleotides resulted in extensive gene expression changes, including upregulation of mitochondrial and metabolism-associated pathways. Together, these results show that differential editing of microRNAs may re-direct targeting and result in altered functions relevant to the pathophysiology of temporal lobe epilepsy and perhaps other disorders of neuronal hyperexcitability.

3.
Ther Adv Med Oncol ; 12: 1758835919897546, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32064003

RESUMO

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Epidermal growth factor receptor (EGFR) has been shown to be over-expressed in TNBC and represents a rational treatment target. METHODS: We examined single agent and combination effects for afatinib and dasatinib in TNBC. We then determined IC50 and combination index values using Calcusyn. Functional analysis of single and combination treatments was performed using reverse phase protein array and cell cycle analysis. Finally, we determined the anticancer effects of the combination in vivo. RESULTS: A total of 14 TNBC cell lines responded to afatinib with IC50 values ranging from 0.008 to 5.0 µM. Three cell lines, belonging to the basal-like subtype of TNBC, were sensitive to afatinib. The addition of afatinib enhanced response to the five other targeted therapies in HCC1937 and HDQP1 cells. The combination of afatinib with dasatinib caused the greatest growth inhibition in both cell lines. The afatinib/dasatinib combination was synergistic and/or additive in 13/14 TNBC cell lines. Combined afatinib/dasatinib treatment induced G1 cell cycle arrest. Reverse phase protein array results showed the afatinib/dasatinib combination resulted in efficient inhibition of both pERK(T202/T204) and pAkt(S473) signalling in BT20 cells, which was associated with the greatest antiproliferative effects. High baseline levels of pSrc(Y416) and pMAPK(p38) correlated with sensitivity to afatinib, whereas low levels of B-cell lymphoma 2 (Bcl2) and mammalian target of rapamycin (mTOR) correlated with synergistic growth inhibition by combined afatinib and dasatinib treatment. In vivo, the combination treatment inhibited tumour growth in a HCC1806 xenograft model. CONCLUSIONS: We demonstrate that afatinib combined with dasatinib has potential clinical activity in TNBC but warrants further preclinical investigation.

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