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1.
Cancer Biol Med ; 2024 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-38940672

RESUMEN

OBJECTIVE: Radiotherapy has achieved remarkable effects in treating non-small cell lung cancer (NSCLC). However, radioresistance remains the major obstacle to achieving good outcomes. This study aims at identifying potential targets for radiosensitizing NSCLC and elucidating the underlying mechanisms. METHODS: Lentivirus-based infection and CRISPR/Cas9 technology were used to modulate the expression of microRNA-384 (miR-384). Cell clonogenic formation assays and a xenograft tumor model were used to analyze radiosensitivity in NSCLC cells. Fluorescence-activated cell sorting was used to assess the cell cycle and cell death. Immunofluorescence staining, Comet assays, and homologous recombination or non-homologous end-joining I-SceI/GFP reporter assays were used to study DNA damage and repair. Western blotting and quantitative real-time polymerase chain reaction were used to identify the targets of miR-384. Chromatin immunoprecipitation and polymerase chain reaction were performed to evaluate upstream regulators of miR-384. RESULTS: MiR-384 was downregulated in NSCLC. Overexpression of miR-384 increased the radiosensitivity of NSCLC cells in vitro and in vivo, whereas knockout of miR-384 led to radioresistance. Upregulation of miR-384 radiosensitized NSCLC cells by decreasing G2/M cell cycle arrest, inhibiting DNA damage repair, and consequently increasing cell death; miR-384 depletion had the opposite effects. Further investigation revealed that ATM, Ku70, and Ku80 were direct targets of miR-384. Moreover, miR-384 was repressed by NF-κB. CONCLUSIONS: MiR-384 is an ionizing radiation-responsive gene repressed by NF-κB. MiR-384 enhances the radiosensitivity of NSCLC cells via targeting ATM, Ku80, and Ku70, which impairs DNA damage repair. Therefore, miR-384 may serve as a novel radiosensitizer for NSCLC.

2.
EMBO Mol Med ; 16(4): 1027-1045, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38448545

RESUMEN

Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXOEAA-PMO). EXOEAA-PMO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXOEAA-PMO elicited therapeutic levels of dystrophin restoration and functional improvements in mdx mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.


Asunto(s)
Exosomas , Distrofia Muscular de Duchenne , Animales , Ratones , Distrofina/genética , Ratones Endogámicos mdx , Exosomas/metabolismo , Morfolinos/metabolismo , Morfolinos/farmacología , Morfolinos/uso terapéutico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleótidos/metabolismo , Oligonucleótidos/uso terapéutico
3.
Am J Transl Res ; 14(8): 5466-5479, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36105026

RESUMEN

OBJECTIVES: To validate that dexlansoprazole, an anti-acid drug, can prevent pulmonary artery hypertension (PAH) in preclinical animal models and find the possible mechanism of action of dexlansoprazole for this new indication. METHODS: The efficacy of dexlansoprazole to attenuate PAH in vivo was evaluated in PAH animal models. Plasma guanosine 3', 5'-cyclic phosphate (cGMP) in PAH rats was measured by enzyme linked immunosorbent assay (ELISA). To investigate the anti-PAH effect of dexlansoprazole in vitro, proliferation and migration assays of primary cultured pulmonary artery smooth muscle cells (PASMCs) were performed. Furthermore, dexlansoprazole's function on fibroblast transition of vascular smooth muscle cells (VSMC) was explored by single cell ribonucleic acid (RNA) sequencing and RNAscope. RESULTS: Dexlansoprazole could attenuate the pathologic process in monocrotaline (MCT)-, hypoxia-induced PAH rats and SU5416/hypoxia (SuHy)-induced PAH mice. The intervention with dexlansoprazole significantly inhibited elevated right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and pulmonary vascular wall thickness. Furthermore, plasma cGMP in MCT-induced PAH rats was restored after receiving dexlansoprazole. In vitro, dexlansoprazole could inhibit PASMCs' proliferation and migration stimulated by platelet derived growth factor-BB (PDGF-BB). Moreover, dexlansoprazole significantly ameliorated pulmonary vascular remodeling by inhibiting VSMC phenotypic transition to fibroblast-like cells in a VSMC-specific multispectral lineage-tracing mouse. CONCLUSIONS: Dexlansoprazole can prevent PAH through promoting cGMP generation and inhibiting pulmonary vascular remodeling through restraining PASMCs' proliferation, migration, and phenotypic transition to fibroblast-like cells. Consequently, PAH might be a new indication for dexlansoprazole.

4.
Hypertension ; 79(6): 1203-1215, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35354317

RESUMEN

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotype transition plays an essential role in vascular remodeling. PGD2 (Prostaglandin D2) is involved in cardiovascular inflammation. In this study, we aimed to investigates the role of DP1 (PGD2 receptor 1) on VSMC phenotype transition in vascular remodeling after Ang II (angiotensin II) infusion in mice. METHODS: VSMC-specific DP1 knockout mice and DP1flox/flox mice were infused with Ang II for 28 days and systolic blood pressure was measured by noninvasive tail-cuff system. The arterial samples were applied to an unbiased proteome analysis. DP1f/f Myh11 (myosin heavy chain 11) CREERT2 R26mTmG/+ mice were generated for VSMC lineage tracing. Multiple genetic and pharmacological approaches were used to investigate DP1-mediated signaling in phenotypic transition of VSMCs in response to Ang II administration. RESULTS: DP1 knockout promoted vascular media thickness and increased systolic blood pressure after Ang II infusion by impairing Epac (exchange protein directly activated by cAMP)-1-mediated Rap-1 (Ras-related protein 1) activation. The DP1 agonist facilitated the interaction of myocardin-related transcription factor A and G-actin, which subsequently inhibited the VSMC transition to myofibroblasts through the suppression of RhoA (Ras homolog family member A)/ROCK-1 (Rho associated coiled-coil containing protein kinase 1) activity. Moreover, Epac-1 overexpression by lentivirus blocked the progression of vascular fibrosis in DP1 deficient mice in response to Ang II infusion. CONCLUSIONS: Our finding revealed a protective role of DP1 in VSMC switch to myofibroblasts by impairing the phosphorylation of MRTF (myocardin-related transcription factor)-A by ROCK-1 through Epac-1/Rap-1/RhoA pathway and thus inhibited the expression of collagen I, fibronectin, ED-A (extra domain A) fibronectin, and vinculin. Thus, DP1 activation has therapeutic potential for vascular fibrosis in hypertension.


Asunto(s)
Angiotensina II , Hipertensión , Receptores Inmunológicos , Receptores de Prostaglandina , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Células Cultivadas , Fibronectinas/metabolismo , Fibrosis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hipertensión/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Factores de Transcripción/metabolismo , Remodelación Vascular/genética
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