沉默LncRNA GAS5通过miR‑21/PTEN轴抑制高氧诱导的Ⅱ型肺泡上皮细胞凋亡OA

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.