| Objective:Acute lung injury(ALI)/acute respiratory distress syndrome(ARDS)is an acute inflammatory lung injury caused by various pulmonary and extrapulmonary pathogenic factors.In the biological process of ALI/ARDS,microRNA150(miR-150)is considered to be involved in a variety of physiological and pathological processes,including immune and inflammatory response,hematopoietic differentiation and activation of immune cells,which are essential for the development of ALI/ARDS.However,the role of miR-150 in ALI/ARDS has not been widely reported.The aim of this study was to detect the level of miR-150 in the serum of ARDS patients,to explore the effect of miR-150 on type Ⅱ alveolar epithelial cell line A549,and to confirm the actual effect of miR-150 in ALI mouse model by animal experiments.Methods:Part one:The cohort study recruited 120 cases of ARDS patients and 60 cases of age and gender-matched healthy volunteers.The expression level of miR-150 was detected by fluorescent quantitative RT-PCR and the expressions of related cytokines in serum were detected by ELISA assay.The acute physiology and chronic health assessment(APACHE)Ⅱ score,sequential organ failure assessment(SOFA)score and lung injury(LIS)score of each group were recorded accordingly.The baseline characteristics of the two groups were further analyzed,such as age,gender,body mass index(BMI),potential risk factors for ARDS,and the severity of the disease once admission to ICU.All subjects were followed up for 28 days and the 28-day mortality rate was also analyzed.Part two:Anesthetized mice(10 mice in each group)with 2%chloral hydrate(0.2 ml/10 g),and intubated into the trachea through an oral sterile plastic catheter.Acute lung injury model was established by LPS(5 mg/kg)nasal drip for 72 hours,and mice injected with the same dose ofPBS were used as a control group.The mice were injected with agomir-150 and agomir-negative control(agomir-NC)via tail vein injection.Survival of all mice was detected and recorded every day and continuedfor 3 days.Three days later,the lung tissues of each group were taken,and the left lung was embedded in paraffin,sliced at 5 um,and stained with hematoxylin and eosin(HE).Total RNA was extracted from right lung tissues and spleen tissues,and the expressions of inflammation-related IL-1β、IL-6 and TNF-α mRNAs were detected by quantitative RT-PCR.Bronchoalveolar lavage(BAL)fluid was taken from each group,eyeball blood was taken from each group,and serum was collected after centrifugation.The levels of inflammatory factors IL-1β,IL-6 and TNF-α were detected by ELISA,and leukyocyte and neutrophils were counted by flow cytometry.Part three:Liposome transfection was used to transfect miR-150 mimics into A549 cells and up-regulate the expression of miR-150.Lentivirus-mediated AKT3 siRNA interfered with AKT3 expression in human alveolar type Ⅱ epithelial cell line A549.Different concentrations of LPS were used to stimulateA549 cellsin every group.Cells were collected at 6h,12h,24h,36h and 48h for protein extraction and quantification.Western blot was used to detect the expressions of inflammatory factors,autophagy proteins,apoptosis proteins and related signal pathway proteins such as NF-kappa B and JNK.ELISA was used to detect the levels of inflammatory factors in extracted supernatant,Annexin V-FITC/PI double-stained cells were used to detect apoptotic cell rates by flow cytometry,and CCK-8 was used to detect the proliferation capacity of A549 cells.Dual luciferase reporter gene assay was used to verify the direct post-transcriptional regulation relationship of miR-150 and AKT3.Results:Part one:Firstly,a significant reduction in the miR-150 level in serum of patients with ARDS compared with the healthy controls(P<0.01).Specially,the expression level of miR-150 in non-survivors was lower than that in survivors(P<0.01).The secretion levels of IL-1β,IL-6 and TNF-α were significantly higher in serum of patients with ARDS compared with healthy controls(P<0.01).Particularly,non-survivors had significantly higher secretion levels of IL-1β IL-6 and TNF-α than those in survivors(P<0.01).The expression level of miR-150 in serum was inversely associated with the serological levels of IL-1β,IL-6 and TNF-α in patients with ARDS(P<0.001).In addition,we analyzed the correlations of miR-150 with APACHE Ⅱ score,SOFA score,lung injury score,and PaO2/FiO2 in ARDS patients.In the included patients with ARDS,the significantly inverse correlations were identified among miR-150 and APACHE Ⅱ score(r=-0.778,P<0.001),SOFA score(r=-0.654,P=0.001),and lung injury score(r=-0.599,P<0.001),respectively.Further,a markedly positive correlation between miR-150 level and PaO2/FiO2(r=0.809,P<0.001)was also demonstrated.Receiver operating characteristic(ROC)curves revealed that the optimal cut-off value of miR-150 expression for diagnosis of ARDS was 1.220(specificity:96.67%,sensitivity:92.50%),and the area under the curve(AUC)value of miR-150 was obviously higher(0.989;95%CI:0.978-0.999)than that of IL-1β(0.854),IL-6(0.862)or TNF-α(0.937)(P<0.001,Fig.1H).ARDS patients with high miR-150 level had a significantly higher 28-day survival rate compared with those with low miR-150 level(P<0.001).Multivariate Cox proportional hazard regression analysis suggested that in addition to APACHE II scores,SOFA score and cytokines,miR-150 was also an independent risk factor for 28-day survival of ARDS patients(hazard ratio:9.135,95%CI:2.246-37.156;P=0.002).Part two:3 days following LPS treatment,the expression level of miR-150 in BAL fluid was significantly reduced compared with PBS control group(P<0.01).Likewise,miR-150 in peripheral blood and splenocytes was decreased 3 days after treatment in LPS-induced ALI mice(P<0.01,Fig.2A).The expression level of miR-150 in the BAL fluid,peripheral blood and splenocytes of mice with miR-150 over-expression was significantly increased by approximately five fold(P<0.01,Fig.2B).In addition,enforced expression of miR-150 effectively decreased total inflammatory cell count,especially neutrophil,and the secretion of inflammatory cytokines in the BAL fluid of LPS-induced ALI mice(P<0.01).Subsequently,miR-150 significantly decreased the levels of total protein,albumin and IgM in the BAL fluid from LPS-treated ALI mice(P<0.05).Consistently,we observed HE stained lung tissue sections,and found that lung tissues from PBS-treated mice showed normal architectures,while lung tissues from LPS-treated mice exhibited severe edema and neutrophil infiltration.Eventually,on the first day following LPS administration,compared with mice treated with LPS alone,miR-150 obviously prolonged the survival rate of mice with LPS and miR-150 overexpression vector(P<0.01).Further,the statistically significant differences between miR-150 over expression group and three other groupsindicated miR-150 markedly improved the survival rate of ALI mice(P<0.01).Part three:LPS markedly repressed cell viability in a time-dependent manner at 72 h after treatment(P<0.05).After treatment with different concentrations of LPS for 72 h,the expression level of miR-150 was significantly reduced in a dose-dependent manner(P<0.01).When A549 cells were treated withLPS for 72 h,LPS significantly increased apoptosis of A549 cells(P<0.001),reduced Bcl-2 level,and elevated Bax,cleaved-caspase-3 and cleaved-caspase-9 levels compared with PBS control(P<0.01).Additionally,in comparison with PBS control,LPS obviously increased the secretion levels of IL-1β,IL-6,and TNF-α in A549 cells(P<0.001)and the expressions of LC3 Ⅱ/Ⅰand Beclinl(P<0.05).These data indicated that LPS induced A549 cell injury.On the other hand,over-expression of miR-150 obviously alleviated LPS-inhibited A549 cell viability(P<0.01),inhibited LPS-induced apoptosis(P<0.01),reversed the expression levels of apoptosis-related proteins,suppressed the secretion levels of IL-1β,IL-6and TNF-α(P<0.01),and decreased the expressions of LC3 Ⅱ/Ⅰ and Beclinl.TargetScan identified putative target sequences at position 1211-1218 of the AKT33’-UTR,AKT3 was chose as a potential target of miR-150.Western blot assay demonstrated that the level of AKT3 protein was significantly decreased in A549 cells transfected with miR-150 mimics compared with miR-NC(P<0.01).Subsequently,luciferase reporter assay was further used to confirm the predicted target of miR-150.We demonstrated that miR-150 mimics significantly decreased the relative luciferase activity in HEK-293T cells co-transfected with AKT3 WT 3’-UTR reporter compared with miR-NC(P<0.01).Nevertheless,the relative luciferase activity in HEK-293T cells with AKT3 MUT 3’-UTR was not altered P>0.05).To elucidate the regulatory relationship between miR-150 and AKT3,we inhibited the AKT3 in the presence of LPS,silencing of AKT3 inhibited release of inflammatory factor of A549 cell(P<0.01),attenuated cell apoptosis(P<0.01)via increasing the level of Bcl-2 and decreasing the levels of Bax,cleaved-caspase-3 and cleaved-caspase-9(P<0.01).In addition,silencing of AKT3 inhibited the production of IL-1β,IL-6,and TNF-α(P<0.01)and autophagy through reducing the expressions of LC3 II/I and Beclinl.Notably,miR-150 mimics together with AKT3 silencing synergically protected A549 cells against LPS-induced cell injury than three other groups(P<0.01).In comparison with LPS alone,the expression levels of p-JNK,p-p65,and p-IκBαwere obviously decreased by miR-150 mimics or si-AKT3 in the presence of LPS(P<0.01).Most importantly,the inhibitory effects of miR-150 mimics alone on the expressions of p-JNK,p-p65,and p-IκBα were greatly aggravated by silencing of AKT3(P<0.01).Conclusions:MiR-150 has clinical significance in early diagnosis and prognosis of ARDS patients.MiR-150 can alleviate lung inflammation in ALI mice,and can protect ALI mice and promote its survival.MiR-150 inhibits JNK and NF-κB pathways by directly targeting AKT3,and protects A549 cells against LPS-induced inflammatory injury,autophagy and apoptosis.MiR-150 may be a potential target for the treatment of acute lung injury/acute respiratory distress syndrome. |
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