MicroRNA-21-5p Activates PI3K/AKT/mTOR Pathway By PTEN To Regulate H₂O₂-induced Autophagy And Apoptosis In AECⅡ

Objective:The phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin（PI3K/AKT/m TOR）signaling pathway plays an important role in the regulation of autophagy,and activation of the PI3K/AKT/m TOR pathway can inhibit the occurrence of autophagy.The relationship between PI3K/AKT/m TOR pathway and hyperoxia-induced type II alveolar epithelial cells（AECII）autophagy is still unclear.This study aims to explore the mechanism of micro RNA-21-5p（miR-21-5p）activates PI3K/AKT/m TOR pathway by Phosphataseand tensin homolog deleted on chromosome 10（PTEN）to inhibit AEC II autophagy.Methods:After 48 hours of isolation,purification and culture,AECⅡof healthy male SD rats were randomly divided into 9 groups for model construction:group A（Control group:normal culture）,group B（H₂O₂group:0.5mmol/LH₂O₂）,group C（miR-21-5p+H₂O₂group:miR-21-5p lentivirus overexpression vector infection+0.5mmol/LH₂O₂）,group D（Empty vector+H₂O₂group:Empty lentivirus vector infection+0.5mmol/L H₂O₂）,group E（Rapa+H₂O₂group:Rapamycin+0.5mmol/LH₂O₂）,group F（Rapa+miR-21-5p+H₂O₂group:Rapamycin+miR-21-5p lentivirus overexpression vector infection+0.5mmol/LH₂O₂group）,group G（MHY1485+H₂O₂group:MHY1485+0.5mmol/L H₂O₂group）,group H（MHY1485+miR-21-5p+H₂O₂group:MHY1485+miR-21-5p lentivirus silence vector infection+0.5mmol/L H₂O₂group）,group I（DM SO+H₂O₂group:0.02%DMSO+0.5mmol/LH₂O₂group）.The growth morphology of AEC II was observed by inverted phase contrast microscopy;Immunofluorescence staining（IF）was used to observe Surfactant protein C（SP-C）expression by fluorescence microscopy;the characteristic structure of AEC II and autophagy-related bodies were identified by transmission electron microscopy（TEM）;the proliferation activity of AEC II was detected by CCK-8;miR-21-5p expression level was detected by real-time fluorescence quantitative PCR（RT-q PCR）;the purity,efficiency of lentivirus infection,reactive oxygen species（ROS）and early apoptosis of AEC II were detected by flow cytometry（FCM）.Autophagy dual-label system was used to evaluate autophagic flow;the expression of PTEN,p-ATK,m TOR,p-m TOR,LC3and p62 was detected by Western blot.Results:1.Cell morphology:The cells are observed by an optical microscope.At the 24h time point,AECⅡshowed adherent and island-like growth,and the fusion rate at the48h time point was greater than 95%.The unique ultrastructural osmophilic lamellar bodies and microvilli of AECⅡwere observed by transmission electron microscope.After staining SP-C,it was observed by immunofluorescence microscope and AECⅡhad SP-C expression.The purity of AECⅡis 94.77±0.14%.The efficiency of lentivirus infection is over 95%.2.Identification of injury model:H₂O₂can induce the dynamic changes of LC3and p62.Compared with group A,the cell viability of group B decreased significantly.Compared with group A,the production of ROS in group B was significantly increased.Compared with group A,autophagosomes and autophagolysosomes in AECⅡwere significantly increased in the group B（all P<0.05）.3.Determination of the expression level of miR-21-5p:Compared with group A,the expression level of miR-21-5p in group B was significantly down-regulated,and the expression level of miR-21-5p in group C was significantly increased（all P<0.05）.4.CCK-8:Compared with group A,the AECⅡviability of group B was significantly decreased.Compared with group B,the viability of AECⅡin group C was significantly higher（all P<0.05）.5.Detection of PTEN/AKT/m TOR related proteins and LC3 and p62:Compared with group A,p-Akt,p62,LC3-Ⅱ/I were up-regulated in group B,while PTEN and p-m TOR were down-regulated.Compared with group B,p-Akt and p-m TOR were up-regulated in group C,while PTEN,p62,and LC3-Ⅱ/I were down-regulated（all P<0.05）.6.The exploration of the concentration of m TOR inhibitor and activator:（1）Rapamycin:All concentrations did not affect the viability of AECⅡ.The inhibition of m TOR was significant after 20n M treatment（P<0.05）.（2）MHY1485:All concentrations did not affect the viability of AECⅡ,but cell viability is related to the treatment time of the MHY1485.The activation of m TOR was significant when treated with 2μM for 4h（P<0.05）.7.The expression of m TOR,p-m TOR,LC3 and p62 in each group after treatment with m TOR inhibitor:Compared with group B,the expression of p-m TOR and p62 in group E was down-regulated,and the expression of LC3-Ⅱ/Ⅰwas up-regulated.Compared with group E,the expression of LC3-Ⅱ/I in group F was down-regulated（all P<0.05）.8.The expression of m TOR,p-m TOR,LC3 and p62 in each group after treatment with m TOR activator:Compared with group B,the expression of p-m TOR and p62 in the group G was up-regulated,and the expression of LC3-Ⅱ/I was down-regulated.Compared with group G,the expression of p62 in group H was down-regulated（all P<0.05）.9.The early rate of apoptosis of each group was measured by flow cytometry:Compared with group A,the early rate of apoptosis of AECⅡin group B was significantly increased.Compared with group B,the early rate of apoptosis of AECⅡin group C was significantly decreased,and the early rate of apoptosis of AECⅡin the group E was decreased,and the early rate of apoptosis of AECⅡin group G was increased.Compared with the group C,the early rate of apoptosis of AECⅡin the group F was increased,and the early rate of apoptosis of AECⅡin the group H was increased（all P<0.05）.10.Evaluation of autophagic flux:Compared with group A,the number of autophagosomes in AECⅡof group C were significantly decreased,and the number of autophagosomes and autophagolysosomes in AECⅡof group E were significantly increased,and the number of autophagosomes and autophagolysosomes in AECⅡof group G were significantly decreased.Compared with the group C,the number of autophagosomes and autophagolysosomes of group F were significantly increased,and the number of autophagosomes and autolysosomes of group H was significantly increased（all P<0.05）.Conclusion:1.AECⅡtreated with H₂O₂can induce autophagy and apoptosis;2.miR-21-5p inhibits the autophagy and apoptosis of AECⅡby targeting PTEN to activate PI3K/AKT/m TOR signaling pathway.