Objective To investigate the mechanism of circRNA_0001454 carried by extracellular vesicles (EV) derived from umbilical cord mesenchymal stem cells (MSC) on regulation of autophagy in alveolar epithelial cells in acute respiratory distress syndrome (ARDS).
Methods MSC were isolated and cultured from the umbilical cords of full-term neonates born between January and December 2024 at the Second Affiliated Hospital of Xinjiang Medical University using the tissue explant adherent method and subsequently identified. EV were isolated by ultracentrifugation and identified using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Human pulmonary alveolar epithelialcells (HPAEpiC) were selected for the experiment, and an in vitro ARDS cell model was established by stimulating HPAEpiC with lipopolysaccharide for 12 h. The experiment was divided into 6 groups: control group, MSC-EV+model group (MSC-EV group), model group, empty vector group, overexpression group, and low-expression group. Except for the control group, models were replicated in all other groups. In the empty vector, overexpression and low-expression groups, HPAEpiC were co-cultured for 24 h with MSC-EV carrying an empty vector, circRNA_0001454 overexpression plasmid vector, or low-expression plasmid vector, respectively. The uptake of MSC-EV by HPAEpiC was observed using laser confocal microscopy. The expression levels of circRNA_0001454 and miR-9-5p were detected by qRT-PCR. The protein level of silent information regulator 1 (SIRT1) was detected by Western blot. Cell viability was assessed using CCK-8. Apoptosis was detected by TUNEL staining. The formation of autophagosomes was observed by transmission electron microscopy. The ratios of microtubule-associated protein 1 lightchain 3 (LC3)-Ⅱ to LC3-Ⅰ (LC3-Ⅱ/LC3-Ⅰ), Beclin-1, and autophagy-related protein 5 (ATG5) were detected by Western blot. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 levels in the supernatant were detected by ELISA. The targeting relationship between circRNA_0001454 and miR-9-5p was verified through bioinformatics and dual-luciferase reporter assays.
Results MSC and EV were successfully isolated and identified. HPAEpiC successfully took up MSC-EV. The expression level of circRNA_0001454 in HPAEpiC in the MSC-EV group was increased compared with that in the control group, while there was no significant difference between the model group and the control group. The expression levels of miR-9-5p and SIRT1 protein in HPAEpiC in the overexpression group were significantly lower than those in the empty vector and model groups, while the expression levels of miR-9-5p and SIRT1 protein in the low-expression group were significantly higher than those in the empty vector and model groups (P < 0.05), suggesting that circRNA_0001454 can inhibit the expression of miR-9-5p and SIRT1 protein. Compared with the empty vector and model groups, the overexpression group showed significantly increased cell viability and significantly decreased apoptosis rate, autophagosome formation, LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and ATG5 expression, and TNF-α, IL-1β and IL-6 levels. In contrast, the low-expression group showed significantly decreased cell viability and significantly increased apoptosis rate, autophagosome formation, LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and ATG5 expression, and TNF-α, IL-1β and IL-6 levels compared with the empty vector and model groups (P < 0.05). Bioinformatics revealed binding sites between circRNA_0001454 and miR-9-5p, and dual-luciferase reporter assay results and PCR showed that circRNA_0001454 targeted and regulated the expressionof miR-9-5p.
Conclusion CircRNA_0001454 carried by MSC-EV may alleviate autophagy in alveolar epithelial cells in ARDS by adsorbing miR-9-5p and inhibiting the SIRT1 pathway.
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