The Role Of MiRNA-1246 In LPS Induced Acute Lung Injury And Its Related Mechanism

Background and Objective:Acute lung injury(ALI)is a common clinical syndrome characterized by progressive and intractable hypoxemia.When the disease progresses to severe,and oxygenation index <200,it is called acute respiratory distress syndrome(ARDS).Treatment modalities for patients with ALI/ARDS involves general supportive care prescribed for all critically ill patients(e.g.,infection control,nutritional support with an aim for early enteral feeding,good glycaemic control,stress ulceration prophylaxis,and deep venous thrombosis)combined with focused ventilator strategies.These techniques have improved the prognosis of ALI/ARDS to a certain extent.But overall,the effect is not satisfactory,and the ALI/ARDS mortality rate is still above 40%.Therefore,there is an urgent need for further research on the molecular mechanisms of acute lung injury to help develop new treatment options.It is currently believed that ALI/ARDS is caused by excessive and uncontrolled systemic inflammatory response caused by direct or indirect lung injury,and its main pathophysiological features are migration of inflammatory cells,activation of fibroproliferation and apoptosis.Therefore,controlling the "cascade" reaction of inflammation in the lung,preventing the expansion of the inflammatory cascade,and reducing the apoptosis of alveolar epithelial cells are effective measures for the treatment of ALI/ARDS.MicroRNAs(miRNAs)can participate in a series of life processes,such as participating in the regulation of cell proliferation,differentiation,migration and apoptosis,regulating the body’s inflammatory response,immune function,tumor formation and other responses.Studies have found that certain miRNAs are significantly up-regulated or down-regulated in ALI/ARDS cell apoptosis and inflammatory responses.NF-κB plays an important role in the regulation of the production of pro-inflammatory factors such as TNF-α,IL-1β and IL-6,and Wnt/β-catenin signaling pathway is closely related to cell proliferation and apoptosis.In tumors and other related fields,it has been found that miRNA-1246 regulates Wnt/β-catenin and NF-κB signaling pathways involved in apoptosis and inflammatory processes.Therefore,we hypothesized that miRNA-1246 may mediate ALI/ARDS-induced apoptosis and inflammatory responses via Wnt/β-catenin and NF-κB signaling pathways.Through this experiment,the potential regulation ofmiRNA-1246 on ALI/ARDS and related mechanisms were clarified,which provided a new direction and ideas for the prevention and treatment of ALI/ARDS.Methods:1.Twenty SPF grade C57BL/6 mice(5-6 weeks old,18-20 g)were selected.The acute lung injury model was established by intratracheal instillation of lipopolysaccharide(LPS).After 1 day of modeling,we sacrificed mice and detected the degree of lung injury by HE staining,and the expression of miRNA-1246 in lung tissue by RT-qPCR.2.Human alveolar epithelial cell lines(A549 cells)were cultured in vitro and transfected with miRNA-1246,anti-miRNA-1246 and negative mimic into A549 cells.Then,LPS stimulation was given to simulate the alveolar epithelial cell response in ALI/ARDS.RT-qPCR was performed to verify the expression of miRNA-1246 in each group after transfection.3.The proliferation of each group was detected by MTT assay,and the LDH activity of each group was detected by LDH activity kit.4.Apoptosis was detected by Annexin V-PI double staining and flow cytometry.5.Colorimetric assay was used to detect the activity of apoptotic factor caspase-3/9 in each group.6.Western blot was used to detect the expression of Wnt and β-catenin protein in each group7.Wnt inhibitor were used to observe the cell proliferation and apoptosis,the level of apoptosis factor caspase-3/9,the expression changes of Wnt and beta-catenin protein in each group,and to explore the regulatory role and mechanism of miRNA-1246 in the apoptosis of ALI.8.The levels of inflammatory factors IL-1β,IL-6 and chemokines CCL2 and CCL5 in each group were detected by enzyme-linked immunosorbent assay(ELISA).9.Western blot was used to detect the expression of p-NF-κB and p-IκBα in each group.10.NF-B inhibitor were used to observe levels of IL-1β,IL-6,CCL2,CCL5 and the expression of p-NF-κB and p-IκBα,and to investigate the regulation effect and mechanism of miRNA-1246 in inflammatory response of ALI.Results:1.Lung tissues were collected at 1 day after LPS treatment in mice,and subjectedto H&E staining.LPS-induced ALI group showed severe histopathologic changes,including alveolar wall thickness,pulmonary congestion and pulmonary congestion.2.miRNA-1246 expression was effectively up-regulated in LPS-induced ALI group,compared with the control group.3.In ALI cell model,the expression of miRNA-1246 was effectively increased using miRNA-1246 mimic,compared with the control group.Over-expression of miRNA-1246 effectively inhibited cell proliferation and increased LDH activity in comparison with the control group.However,up-regulation of miRNA-1246 effectively increased cell apoptosis and promoted caspase-3 and caspase-9 activity.4.miRNA-1246 expression was reduced using anti-miRNA-1246 mimic,compared with the control group.Down-regulation of miRNA-1246 effectively increased cell proliferation,inhibited LDH activity,decreased cell apoptosis and inhibited caspase-3 and caspase-9 activity in ALI cell model.5.Over-expression of miRNA-1246 effectively suppressed Wnt and β-catenin protein expression,and the down-regulation of miRNA-1246 effectively induced Wnt and β-catenin protein expression in ALI cell model,compared with the control group.6.Wnt and β-catenin protein expression was effectively inhibited by Wnt inhibitor.At the same time,Wnt inhibitor further inhibited cell proliferation,increased LDH activity,promoted cell apoptosis and increased the activity of caspase-3 and caspase-9 compared with miRNA-1246 group without Wnt inhibitor.7.Over-expression of miRNA-1246 increased IL-1β,IL-6,CCL2 and CCL5 levels and down-regulation of miRNA-1246 decreased IL-1β,IL-6,CCL2 and CCL5 levels in ALI cell model,compared with the control group.8.Up-regulation of miRNA-1246 induced p-NF-κB and suppressed p-IκBαprotein expression.In contrast,down-regulation of miRNA-1246 suppressed p-NF-κB and induced p-IκBα protein expression in ALI cell model,compared with the control group.9.Compared with miRNA-1246 group without inhibitor,the expression of p-NF-κB protein was down-regulated and the expression of p-IκBα protein was up-regulated after the use of NF-κB inhibitor.At the same time,the levels of IL-1β,IL-6,CCL2 and CCL5 decreased after the inhibition of NF-κB.Conclusions:1.The expression of miRNA-1246 is up-regulated in the LPS-induced ALImodel.2.miRNA-1246 may participate in the apoptosis process of LPS-induced ALI by inhibiting Wnt/β-catenin signaling pathway.3.miRNA-1246 may participate in the inflammatory process of LPS-induced ALI through activating NF-κB signaling pathway.