MiR-21-5p Regulates Acute Injury Of Alveolar Epithelial Cells Induced By Lipopolysaccharide Through Targeting SLC16A10

Objective: To investigate the expression patterns of miR-21-5p and solute carrier family16 A member 10(SLC16A10)in a model of Lipopolysaccharide(LPS)-induced type II alveolar epithelial cell injury,and the therapeutic effects of their targeted regulation on Acute lung injury(ALI).Methods:(1)To construct a model of LPS-induced type II alveolar epithelial cell injury,A549 cells were directly stimulated with 10 ug/ml of LPS,phosphate buffer(PBS)equal to LPS was added as control,cells were collected at different time points,RNA and protein were extracted,The expression of interleukin 1 beta(IL-1β)and tumor necrosis factor-α(TNF-α)was measured by real-time quantitative pcr(RT-q PCR)and western blot(WB).(2)The expression of miR-21-5p in the cells at each time point after LPS stimulation was detected by RT-q PCR.(3)Up-regulation of miR-21-5p expression in A549 cells by transfection of miR-21-5p mimic,and detection of IL-1β and TNF-αexpression in each group of cells by RT-q PCR and WB method.(4)The miRDB,Target Scan,miRWalk,Starbase,Tarbase and miR Tarbase databases were used to predict miR-21-5p target genes and take the intersection as the target gene intersection group;the Dis Ge Net database was used to search for sepsis-related genes as the sepsis gene group;The intersection of the two groups was designated as the core gene group,and SLC16A10 was identified as the study target by reviewing relevant literature.SLC16A10-WT and miR-21-5p mimic were cotransfected with 293 T cells for 48 h.The luciferase activity was detected by dual luciferase reporter assay to clarify the regulatory relationship between the two;A549 cells were transfected with miR-21-5p mimic and miR-21-5p inhibitor,RT-q PCR and WB method to detect the expression of SLC16A10 and to clarify the effect of regulation of miR-21-5p on SLC16A10 expression.(5)Inhibition of SLC16A10 expression in A549 cells by si RNA,and after adding LPS stimulation,the expression of IL-1β and TNF-α in A549 cells was detected by q PCR and WB method to clarify the effect of inhibition of SLC16A10 expression on LPS-induced inflammatory injury.(6)A549 cells were transfected with miR-21-5p inhibitor alone or with both miR-21-5p inhibitor and si-SLC16A10,and the changes in the expression of IL-1β,TNF-α after inhibition of miR-21-5p expression alone and simultaneous inhibition of miR-21-5p and SLC16A10 expression were compared.To clarify the role of miR-21-5p targeting SLC16A10 in LPS-induced inflammatory injury of A549 cells.Results:(1)IL-1β and TNF-α mRNA and protein expression were significantly increased at 6,12 and 24 h after LPS(10ug/ml)stimulation in A549 cells relative to the control group(P < 0.05),and mRNA peaked at 6 h,while protein levels were significantly time-dependent,with higher relative expression at 24 h.(2)Compared with the control group,miR-21-5p expression was significantly higher in A549 cells after LPS stimulation(P<0.05),but no significant time correlation was observed.(3)Transfection of miR-21-5p mimic upregulated miR-21-5p expression in A549 cells,and the expression of IL-1β,TNF-α was significantly reduced in cells transfected with miR-21-5p mimic after LPS stimulation compared with the control group(P < 0.05),suggesting that miR-21-5p has a protective effect.(4)The target genes of miR-21-5p were predicted by miRDB,Target Scan,miRWalk,Starbase,Tarbase and miR Tarbase,and the intersection was designated as the target gene intersection group,containing 51 genes;the sepsis-related genes retrieved by Dis Ge Net database were designated as the sepsis gene group,containing 1448 genes.The intersection of the two groups was designated as the core gene group,including SLC16A10,TNPO1,STAT3,PIK3R1,and FASLG,.and the four genes in the core gene group,TNPO1,FASLG,STAT3,and PIK3R1,which had been verified to have a target relationship with miR-21-5p,were excluded by reviewing relevant literature.SLC16A10 was selected as a candidate gene for subsequent experimental studies.Dual luciferase results showed that luciferase activity was significantly inhibited in cells cotransfected with SLC16A10-WT and miR-21-5p mimic compared to those cotransfected with SLC16A10-WT and NC mimic(P < 0.05);in contrast,cells cotransfected with SLC16A10-MUT and miR-21-5p mimic showed essentially unchanged luciferase activity(P>0.05).Transfection with miR-21-5p mimic down-regulated SLC16A10 expression and transfection with miR-21-5p inhibitor up-regulated SLC16A10 expression(P < 0.05),suggesting that SLC16A10 is a target gene of miR-21-5p.(5)Transfection of si-SLC16A10 down-regulated the mRNA and protein levels of SLC16A10 in A549 cells.Compared with A549 cells transfected with NC si RNA,the expression of IL-1β and TNF-α in the former was significantly lower after the same dose of LPS treatment(P<0.05),suggesting that inhibition of SLC16A10 expression could attenuate LPS-induced inflammatory damage.(6)Compared with A549 cells transfected with NC inhibitor,A549 cells transfected with miR-21-5p inhibitor,treated with the same dose of LPS,induced a significant increase in the expression of IL-1β and TNF-α produced(P < 0.05);Compared with A549 cells transfected with miR-21-5p inhibitor only,resulting in a significant compared to elevated transfection of miR-21-5p inhibitor and si-SLC16A10 simultaneously,which significantly reduced IL-1β,TNF-α expression(P<0.05),suggesting that miR-21-5p exerts a protective effect against LPS-induced inflammatory damage through regulating SLC16A10 expression.Conclusions:(1)miR-21-5p is involved in the process of LPS-induced acute inflammatory injury in A549 cells and plays a protective role in it.(2)miR-21-5p attenuated LPS-induced inflammatory response in A549 cells by targeting SLC16A10.(3)SLC16A10 may be a potential target for acute lung injury therapy.