RESUMO
Methods:Cultured human alveolar epithelial A549 cells were assigned into LPS group and blank control group. LPS group was stimulated with LPS and adenosine triphosphate to induce pyroptosis and inflammation. A549 cells were divided into 4 groups: miR-20a mimics group, mimics-negative control (NC) group, inhibitor group and inhibitor-NC group. MiRNA-20a mimics, mimics-NC, inhibitor, and inhibitor-NC were transfected respectively into A549 cells, and after 24 h, the cells were collected to verify transfection efficiency by qPCR. MiRNA-20a mimics and the constructed TLR4-3'UTR double luciferase reporter plasmid were co-transfected into A549 cells, and luciferase activity was analyzed. MiRNA-20a mimics/inhibitors were transfected into A549 cells, and then the cells were stimulated by LPS for 8 h followed by adenosine triphosphate for 30 min. QPCR, Western Blot and ELISA were used to detect the expression of GSDMD, inflammatory factors (ASC, NLRP3, Caspase-1, IL-1β) and Signaling molecules (TLR4、NF-κB) in A549 cells at mRNA level and protein level. Immunofluorescence was used to detect the expression of TLR4 in the A549 cells and NF-κB in the nucleus of A549 cells after transfecting with miRNA-20a mimics/inhibitor.Results:The mRNA and protein expression of pyroptosis marker molecule (GSDMD) and inflammatory factors (ASC, NLRP3, Caspase-1, IL-1β) in A549 cells stimulated with LPS were significantly higher than those in the blank control group, and the differences were statistically significant ( P<0.05). The expression of miRNA-20 in the mimics group was significantly higher than that in the mimic-NC group ( P<0.05), while the expression of miRNA-20a in the inhibitor group was lower than that in the inhibitor-NC group ( P<0.01). The double luciferase reporter gene experiment showed that the relative fluorescence value of the co-transfection group for TLR4-3'UTR-WT and miRNA-20a mimics was significantly lower than the co-transfection group for TLR4-3'UTR-WT and miRNA-20a mimics-NC ( P<0.05). The mRNA and protein levels of pyroptosis marker molecule (GSDMD) , inflammatory factors (ASC, NLRP3, Caspase-1, IL-1β) and signaling molecules (TLR4, NF-κB) were decreased in the mimics group compared to the mimics-NC group, and increased in inhibitor group compared to inhibitor-NC group. Conclusions:miRNA-20a may inhibit LPS-induced pyroptosis and inflammation of A549 cells via TLR4/NF-κB signal pathway.Objetive:To explore the potential role of miRNA-20a in lipopolysaccharide (LPS) induced pyroptosis and inflamation of human alveolar epithelial A549 cells and its regulation mechanisim.
RESUMO
Objective:Differentially expressed circ_Dock6 was screened in vivo by applying circRNA high-throughput sequencing technology in lung tissue of newborn rats suffering from acute respiratory distress syndrome (ARDS). The corresponding target genes of microRNAs were predicted by bioinformatics, and their biological processes and signal pathways were analyzed as well. Methods:Real-time quantitative PCR was utilized to detect the expression of circ_Dock6 in the lung tissue of newborn rats in ARDS group (12 cases) and normal control group (12 cases). TargetScan, RNAhybrid and miRanda databases were adopted to predict the possible recruitment of miRNAs and their corresponding target genes by circ_Dock6.Functional enrichment analysis and signal pathway enrichment analysis were carried out on the target genes of each miRNA.Results:The expression of circ_Dock6 (0.44±0.29) in the lung tissue of ARDS group was significantly down-regulated ( t=2.060, P<0.05) compared with normal control group(1.63±1.33). The target gene intersections of miRNAs (miR-24-3p, miR-667-3p, miR-711, miR-203b-5p, miR-5132-5p, etc.) may be recruited by circ_Dock6 and were obtained from three databases.Its target gene aggregation function was enriched in various biological processes, including protein metabolism, protein amino acid phosphorylation, DNA-dependent transcriptional regulation, biological regulation, tissue and organ development, cell differentiation, signal regulation, gene expression, response to stimuli, almost all cellular components such as intracellular, organelle, cytoplasm, and nucleus, as well as molecular functions such as transferase activity, transcription factor activity, and phosphotransferase activity.The involved signaling pathways, including enrichment in mitogen-activated protein kinase(MAPK) signaling pathway, phosphatidylinositol-3-kinase-protein kinase B(PI3K-Akt)signaling pathway, and mammalian rapamycin target protein(mTOR)signaling pathway, were closely related to ARDS.Circ_Dock6 may play a significant role in the pathogenesis of ARDS. Conclusions:Circ_Dock6 may be closely correlated with the pathogenesis of neonatal ARDS.Through bioinformatics analysis, the prediction of its target genes and related signaling pathways laid the foundation for further explorations of its mechanism of action.
RESUMO
Objective To study the effect of small interfering ribonucleic acid (siRNA) silencing apoptosis signal-regulating kinase 1 (ASK1) on inflammatory response of lipopolysaccharide-induced alveolar epithelial A549 cells and its mechanism.Method Cell inflammation model of A549 cells was induced by lipopolysaccharide.The expression of ASK 1 in A549 cells was silenced by liposome transfection of siRNA.The mRNA and expression levels of ASK1,interleukin 6 (IL-6),interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) in A549 cells were detected by immunoblotting,real-time fluorescence quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.Result The expression of IL-6,IL-8 and TNF-α in the experimental group was significantly higher than that in the control group (P＜0.001),which indicated that the inflammatory model of A549 cells was successfully constructed.The mRNA level and expression of ASK1 in the interference group was significantly lower than that in the negative control group and the blank control group (P＜0.01),indicating that silencing ASK1 was also successful.The expressions of IL-6,IL-8 and TNF-α in the interference group (0.37±0.04,0.32±0.04,0.48 ±0.13) were significantly lower than those in the negative control group (1.04±0.11,1.22±0.19,0.93±0.14) and the blank control group (1.01±0.14,1.01 ±0.23,1.02±0.25).The expression of IL-6,IL-8 and TNF-α protein in the interference group (pg/ml) (122.6± 11.0,537.2±42.4,159.2± 19.6) were also significantly lower than those in the negative control group (267.4±20.4,1 289.8±55.3,327.0±26.3) and blank control group (246.6±18.7,1 300.3±35.6,325.2± 18.3),with significant difference (P＜0.05).There was no significant difference in each value between negative control group and blank control group (P＞0.05).Conclusion Silencing ASK1 by siRNA can down-regulate the expression of IL-6,IL-8 and TNF-α in A549 cells,suggesting that ASK 1 may be involved in the regulation of lipopolysaccharide-induced inflammation in A549 cells.
RESUMO
Objective To investigate the role of signal transducer and activator of transcription 3 (STAT3) on the expression of surfactant proteins (SP) in alveolar epithelial cells line A549.Methods STAT3 overexpression lentivirus vector was constructed and transfected into A549 cells.Three small interfering RNAs (siRNA) were chemically synthesized and transfected into A549 cells by Lipofectamine 3000 to construct cells that silenced STAT3.The expression of STAT3,SP-A,SP-B,SP-C and SP-D were detected by Real-time PCR and Western blot.Results In A549 cells,over-or under-regulation of STAT3 were constructed successfully.When STAT3 was rendered over-expressed,the expression of SP-A,SP-B,SP-C,SP-D mRNA was significantly increased compared with Mock group(P ＜ 0.05).The proteins were found to be significantly increased as well.By contrast,when STAT3 was under-expressed,SP was down-regulated (P ＜ 0.05).Conclusion STAT3 regulates the expression of pulmonary surfactant proteins in A549 cells.Over-expression of the STAT3 gene promotes the expression of SP,and its under-expression inhibited it.