# Cigarette smoke-induced circFOXO3 upregulation enhances autophagy-regulated senescence of type II alveolar cells through interacting with E2F1

## TL;DR

Cigarette smoke increases circFOXO3, which worsens lung cell aging and injury through autophagy and E2F1 interactions.

## Contribution

This study reveals a novel mechanism where circFOXO3 promotes cigarette smoke-induced alveolar cell senescence via autophagy and E2F1.

## Key findings

- circFOXO3 knockdown reduces cigarette smoke-induced senescence in AT-II cells.
- circFOXO3 interacts with E2F1 and suppresses its nuclear translocation.
- circFOXO3 knockdown mitigates autophagy impairment and lung injury in vivo.

## Abstract

Senescence of type II alveolar (AT-II) cells is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have reported that circRNA FOXO3 (circFOXO3) is upregulated after exposure to cigarette smoke (CS) and that circFOXO3 knockdown has protective effects on CS-induced inflammation. Here, we investigate whether circFOXO3 upregulation is involved in CS-induced AT-II cell senescence.

Within this experimental cell-based and animal study, the effects of circFOXO3 on CSE-induced senescence in AT-II cell line MLE12 were determined by senescence-associated β-galactosidase staining and western blotting analyses of p16 and p21 expression. Immunofluorescence staining was used to determine the expression of γ-H2AX to analyze DNA damage. Then the autophagy level of CSE-treated MLE12 cells was evaluated by western blotting analyses of LC3B and Beclin-1 expression. Furthermore, we analyzed interactions between circFOXO3 and E2F transcription factor 1 (E2F1) in RNA binding protein immunoprecipitation studies.

Our results show that circFOXO3 knockdown suppressed CS extract (CSE)-induced senescence in the AT-II cell line MLE-12. Additionally, CSE-induced autophagy impairment was reduced by circFOXO3 knockdown, and the autophagy inhibitor 3-methyladenine abrogated the effects induced by circFOXO3 knockdown on cell senescence. Mechanistic investigations revealed that circFOXO3 interacts with E2F1 and suppresses its nuclear translocation. E2F1 knockdown reduced the positive regulation of circFOXO3 knockdown on autophagy and prevented the suppressive effects of circFOXO3 knockdown on cell senescence. Consistent with this, circFOXO3 knockdown mitigated CS-induced senescence in AT-II cells in vivo.

Overall, these findings suggest that CS-induced circFOXO3 upregulation promotes autophagy-dependent senescence of AT-II cells, leading to enhanced lung injury.