| Objective:This study aims to explore the role of miR-20b in hyperoxia lung injury.We first measured the expression levels of miR-20b in both cellular and animal models,followed by revealing the effects of miR-20b on mitochondrial function and apoptosis of lung epithelial cells.We finally unveiled that miR-20b involved in the pathogenesis of hyperoxia lung injury by directly targeting MFN1/MFN2.Methods:Section one:Male SD rats weighing 200-220g were randomly divided into normal control group and HALI group.The rats in HALI group were placed in an oxygen box with an oxygen concentration of 95%and continuously exposed for 48 h to establish the hyperoxia lung injury model.The lung tissues of rats were collected and subjected to HE,Masson,or TUNEL staining to indicate the pathological changes.Bronchoalveolar lavage fluids were also collected,and TNF-α,IL-6,IL-1β levels were measured by ELISA.The levels of superoxide dismutase(SOD),nitric oxide(NO),malondialdehyde(MDA),catalase(CAT)were also similarly quantified.The activities of Na+-K+-ATPase and Ca2+-Mg2+ATPase in lung tissues were detected by ELISA.RT-qPCR was used to evaluate the expression levels of miR-20b in lung tissues.Section two:AECⅡ cells were divided into two groups:control group and H2O2 group(cells were treated with 500 μM H2O2 for 48 h).The ROS levels and apoptotic rates were detected via flow cytometry;the mitochondrial membrane potential was detected by JC-1 staining;and the expression levels of miR-20b were detected by both fluorescence in situ hybridization and RT-qPCR.AEC Ⅱ cells were transfected with miR-20b or NC mimic,respectively,followed by treatment with 500 μM H2O2 for 48 h.These samples were divided into NC mimic group,H2O2+NC mimic group and H2O2+miR-20b mimic group.The expression levels of miR-20b,the ROS level,mitochondrial membrane potentials,and the apoptotic rates were compared among these three groups.Section three:The potential target gene of miR-20b was predicted using TargetScan,and the regulatory relationship between miR-20b and MFN1/MFN2 gene was verified using dual-luciferase reporter assay.AEC Ⅱ cells were divided into four groups.NC mimic,miR20b mimic,NC inhibitor,or miR-20b inhibitor were transfected,respectively.The effects of miR-20b on mRNA and protein expression of MFN1/MFN2 were detected by RT-qPCR and Western blot,respectively.The protein level of MFN1 and MFN2 in AEC II cells after hyperoxia injury were detected by Western blot.The rescue experiment to verify the protective role of miR-20b by targeting MFN1/MFN2 was performed by dividing cells into H2O2 group,H2O2+miR-20b mimic group,H2O2+miR-20b mimic+MFN1 OE group,and H2O2+miR-20b mimic+MFN2 OE group.The protein levels of MFN1 and MFN2,cleaved PARP,and cleaved caspase-3 in each group were detected by Western blot.Cellular apoptosis was detected by flow cytometry.The animal model of hyperoxia lung injury to verify the role of miR-20b in regulating MFN1 and MFN2 to reduce lung injury was constructed by dividing rats were into hyperoxia group,hyperoxia+miR-20b mimic group,hyperoxia+miR-20b mimic+MFN1 OE group and hyperoxia+miR-20b mimic+MFN2 OE group.The protein levels of MFN1 and MFN2,cleaved PARP,and cleaved caspase-3 in each group were detected by Western blot.The pathological changes of lung tissues in each group were assessed by HE staining.Results:Section one:Compared with the normal control group,HE staining of lung tissues in the HALI group showed alveolar structural disorder,alveolar rupture,alveolar wall edema,incomplete alveolar septum,inflammatory cell infiltration in alveoli and stroma.Masson staining showed that the fibrosis level of lung tissues in the HALI group significantly increased.TUNEL staining showed significantly increased apoptosis of lung tissues in the HALI group.Compared with the normal control group,TNF-α,LL-6,IL-1β in alveolar lavage fluids in the HALI group significantly up-regulated(P<0.01).In the HALI group,the levels of antioxidant factors SOD and CAT in alveolar lavage fluid decreased significantly(P<0.01),while the levels of MDA and NO increased significantly(P<0.01).ELISA results showed that the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in HALI group were significantly lower than those in the normal control group(P<0.01).Compared with the normal control group,the expression level of miR-20b in the lung tissues of rats in the HALI group markedly decreased by 66%(P<0.01).Section two:Compared with the normal control group,the ROS level in H2O2 group increased significantly,whereas the mitochondrial membrane potential decreased significantly(P<0.01),and the apoptosis rate of AEC Ⅱ cells increased significantly(29.69%vs 11.81%,P<0.01).Compared with the normal control group,the expression level of miR20b in H2O2 group significantly downregulated by 43%(P<0.01).Compared with NC mimic group,the expression level of miR-20b in H2O2+NC mimic group decreased significantly by 65%(P<0.01).After miR-20b overexpression,compared with H2O2+NC mic group,the expression level of miR-20b in H2O2+miR-20b mic group significantly increased by 115 times(P<0.01).Compared with the NC mimic group,the mitochondrial membrane potential of the H2O2+NC mimic group decreased significantly,whereas the ROS level increased,and the apoptosis rate increased from 14.31%to 34.78%(P<0.01).Compared with the H2O2+NC mimic group,the mitochondrial membrane potential of the H2O2+miR-20b mimic group increased significantly,whereas the level of intracellular ROS decreased significantly,and the apoptosis rate decreased from 34.78%to 18.43%(P<0.01).Section three:TargetScan database prediction showed that miR-20b could reliably bind to the 3’-UTR of MFN1 and MFN2 3’-UTR.Luciferase reporter assay showed that miR-20b directly targeted MFN1 and MFN2.Compared with the NC mimic group,upregulation of miR-20b significantly reduced the expression level of MFN1 mRNA by 74%(P<0.01)and MFN2 mRNA by 78%(P<0.01).Compared with the NC inhibitor group,inhibiting the expression of miR-20b significantly increased the expression of MFN1 mRNA by 2.24 times(P<0.01)and MFN2 mRNA by 2.58 times(P<0.01),respectively.Western blot results showed that,compared with the NC mimic group,miR-20b overexpression could significantly reduce the protein levels of MFN1 by 54%(P<0.01)and MFN2 by 51%(P<0.01).Compared with the NC inhibitor group,miR-20b inhibition significantly increased the protein levels of MFN1 by 2.46 times(P<0.01)and MFN2 by 2.49 times(P<0.01).These results showed that miR-20b could negatively regulate the expression levels of MFN1 and MFN2 in AEC Ⅱ cells.Western blot showed that compared with the normal control group,the expression of MFN 1 and MFN2 protein in H2O2 group increased significantly,indicating that hyperoxia injury can induce the overexpression of MFN1 and MFN2.The rescue experiment results showed that the expression levels of MFN1 and MFN2 in oxidative damaged cells decreased significantly by 44%and 52%(P<0.01),respectively,after transfection with miR-20b mimic.After cotransfection with MFN1 plasmid or MFN2 plasmid,the protein levels of MFN1 and MFN2 increased by 1.63 times and 1.65 times,respectively,indicating that overexpression of MFN1 and MFN2 can counteract the inhibitory and regulatory effects of miR-20b on MFN1 and MFN2.Transfection of miR-20b mimic significantly decreased the expression levels of cleaved PARP and cleaved caspase-3 by 58%and 52%(P<0.01),respectively,and the apoptosis rates decreased to 20%(P<0.01).After cotransfection with MFN1 plasmid,the expression levels of cleaved PARP and cleaved caspase-3 increased significantly by 2.05 and 1.5 times(P<0.01),respectively,and the apoptosis rates increased to 43.03%(P<0.01).After cotransfection with MFN2 plasmid,the expression levels of cleaved PARP and cleaved caspase-3 increased significantly by 2.1 and 1.63 times(P<0.01),respectively.The apoptosis rates increased to 43.09%(P<0.01).These results showed that overexpression of MFN1 or MFN2 could aggravate the apoptosis of oxidatively damaged AECⅡ cells and reverse the protective effects of miR-20b.The data collected in animal models verified that miR-20b played a protective role after hyperoxia lung injury by regulating MFN1/MFN2.Compared with the hyperoxia group,the expression levels of MFN1 and MFN2 in hyperoxia+miR-20b mimic group decreased significantly(P<0.001).Compared with the hyperoxia+miR-20b mimic group,the protein levels of MFN1 in hyperoxia+miR-20b mimic+MFN1 OE group and MFN2 in hyperoxia+miR-20b mimic+MFN2 OE group increased significantly(P<0.001).These results demonstrated that overexpression of MFN1 and MFN2 could counteract the effects of miR20b.Compared with the hyperoxia group,the expression levels of cleaved PARP and cleaved caspase-3 in lung tissues of rats in H2O2+miR-20b mimic group decreased significantly.The expression levels of cleaved PARP and cleaved caspase-3 increased significantly in the hyperoxia+miR-20b mimic+MFN1 OE group and hyperoxia+miR-20b mimic+MFN2 OE group(P<0.001).HE staining showed that in the hyperoxia group,there were a large number of red blood cells exuding from alveolar interstitium,moderate inflammatory infiltration,thickening of alveolar wall and partial expansion of alveolar cavity.In the hyperoxia+miR-20b mimic group,there was only a small amount of red blood cell exudation in local parts,and there was no significant inflammatory infiltration.The alveolar structure was basically complete.In the hyperoxia+miR-20b mimic+MFN1 OE group and hyperoxia+miR-20b mimic+MFN2 OE group,there were some red blood cells exuding from alveolar stroma and mild inflammatory infiltrates.The above results demonstrated that overexpression of MFN1 or MFN2 could aggravate the apoptosis of oxidative injury lung tissue and reverse the protective effect of miR-20b.Conclusion:1.Hyperoxia exposure can aggravate the pulmonary inflammatory response,weaken the antioxidant capacity,reduce the activity of mitochondrial enzymes,and induce apoptosis.The decreased expression of miR-20b in HALI model is closely related to the occurrence and development of hyperoxia lung injury.2.miR-20b can reduce ROS level,increase mitochondrial membrane potential,inhibit apoptosis,and alleviate hyperoxia induced lung injury.3.The expression of MFN1 and MFN2 increase significantly in hyperoxia injury model,and induce caspase-dependent apoptosis.miR-20b inhibits mitochondrial-mediated apoptosis by targeting MFN1 and MFN2,and reduces hyperoxia lung injury.4.miR-20b may become a potential target for the treatment of HALI. |
