Abstract:

Objective To investigate whether long non-coding RNA growth arrest-specific 5 （LncRNA GAS5， GAS5） regulates hyperoxia-induced apoptosis of type Ⅱ alveolar epithelial cells via the microRNA-21-5p （miR-21-5p）/PTEN axis. Methods A hyperoxia-induced acute lung injury （HALI） cell model was established using the murine lung epithelial-12 （MLE-12） mouse type II alveolar epithelial cell line in the logarithmic growth phase， and cells were allocated into four experimental groups： The normoxic control （Control） group， the hyperoxic HALI （HALI） group， the hyperoxia + GAS5 silencing （siGAS5） group， and the hyperoxia + GAS5 silencing + miR-21 silencing （siGAS5 + shmiR-21） group. The following assays were performed in this study： a dual-luciferase reporter gene assay was conducted to verify the direct interaction between GAS5 and miR-21； cell viability was measured via the Cell Counting Kit-8 （CCK-8） assay； the transcript levels of GAS5 and miR-21 were quantified by reverse transcription polymerase chain reaction （RT-PCR）； the levels of superoxide dismutase （SOD） and malondialdehyde （MDA） were determined using commercially available detection kits； reactive oxygen species （ROS） accumulation and mitochondrial membrane potential were observed via immunofluorescence microscopy； the secretion levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6） and interleukin-1β （IL-1β） were measured by enzyme-linked immunosorbent assay （ELISA）； cell apoptosis rate was analyzed via flow cytometry； and the protein expression levels of PTEN， AKT， p-AKT， Cleaved-caspase-3， Bcl-2 and Bax were detected via western blotting. Results GAS5 directly targets and regulates the expression of microRNA-21 （miR-21）. Relative to the control group， the HALI group exhibited significantly decreased cell viability， alongside increased levels of GAS5， MDA， ROS， inflammatory mediators including TNF-α， IL-6， and IL-1β， and an elevated apoptosis rate. Furthermore， the HALI group showed significantly upregulated expression of PTEN， Cleaved caspase-3， and Bax proteins （all P < 0.05）， along with decreased miR-21 expression， reduced SOD activity， lower mitochondrial membrane potential， and significantly downregulated expression of p-AKT and Bcl-2 proteins （all P < 0.05）. Compared with the HALI group， the siGAS5 group had restored cell viability， decreased levels of GAS5， MDA， ROS， inflammatory mediators （TNF-α， IL-6， IL-1β）， and reduced apoptosis rate. The siGAS5 group also showed significantly downregulated expression of PTEN， Cleaved caspase-3， and Bax proteins （all P < 0.05）， along with increased miR-21 expression， elevated SOD activity， higher mitochondrial membrane potential， and significantly upregulated expression of p-AKT and Bcl-2 proteins （all P < 0.05）. Finally， co-silencing of miR-21 largely reversed the cytoprotective effect of GAS5 silencing in MLE-12 cells. Conclusion Silencing of GAS5 attenuates hyperoxia-induced apoptosis in type II alveolar epithelial cells， and this protective effect is potentially mediated via the miR-21/PTEN axis.

Key words: long non-coding RNA GAS5, microRNA-21-5p, apoptosis, type Ⅱ alveolar epithelial cells, hyperoxia-induced acute lung injury