OBJECTIVE  To investigate the mechanism by which circSEPTIN2 competitively binds to miR-144 during autophagy in alveolar epithelial cells （A549） infected with Klebsiella pneumoniae （KPN）.

METHODS  A stable transfection model of KPN-infected A549 cells was developed, with overexpression of circSEPTIN2 and modulation of miR-144 levels. Reverse transcription quantitative real-time PCR （qRT-PCR）, Western blot and fluorescence microscopy were employed to assess the expression levels of miR-144, circSEPTIN2, ARID1A, Notch1 and LC3. A549 cells were infected with KPN standard strains at different multiplicities of infection （MOI） to establish in vitro infection models. Lentiviral transfection was employed to construct cell lines stably overexpressing circSEPTIN2 and modulating miR-144. qRT-PCR and Western blot were performed to measure gene transcription and protein expression, respectively. Dual-luciferase reporter assays were conducted to validate targeting relationships.

RESULTS  KPN infection downregulated circSEPTIN2 and ARID1A expression while upregulating miR-144 in A549 cells. Overexpression of circSEPTIN2 in KPN-infected A549 cells reduced Notch1 and LC3 protein levels, indicating its inhibitory effect on KPN-induced autophagy. Overexpression of miR-144 decreased circSEPTIN2 and ARID1A levels and increased Notch1 and LC3 protein expression, whereas miR-144 inhibition produced the opposite effects. Co-transfection experiments of circSEPTIN2 and miR-144 further confirmed their antagonistic relationship in autophagy regulation: simultaneous overexpression of circSEPTIN2 and inhibition of miR-144 significantly enhanced ARID1A expression and downregulated Notch1 and LC3 levels, demonstrating the strongest autophagy inhibition.

CONCLUSION  circSEPTIN2 and miR-144 play critical roles in regulating autophagy in KPN-infected A549 cells, which provides potential for developing therapeutic strategies targeting these genes.