There is an oral presentation of the scholarly study at 2017 Congress of Asian Pacific Society of Respirology, named THE ROLE OF PM2.5 PLAYED IN CIGARETTE SMOKEINFLAMED PULMONARY EPITHELIUM. Footnotes Disclosure The authors report no conflicts appealing within this work.. involved regulator. Materials and methods Human bronchial epithelial cells (HBEpiCs) were treated with normal media, cigarette smoke solution (CSS) and PM2.5-CSS for 24 h. miR-194-3p mimics, inhibitors and scrambled controls were non-transfected or pre-transfected into HBEpiCs for 48 h. MircroRNAs and mRNA expression were quantified by qRT-PCR. Protein expression was analyzed by western blotting. Caspase activities, mitochondrial membrane potential and TUNEL-positive cells were detected to analyze apoptosis. Bioinformatics and luciferase analysis were used to identify the predicted binding site of miR-194-3p and potential targets. Results In our study, we found that PM2.5 significantly aggravated apoptosis in cigarette-inflamed HBEpiCs. miR-194-3p was dramatically downregulated in PM2.5-CSS-treated HBEpiCs. Bioinformatics and luciferase experiments reported that death-associated protein kinase 1 (DAPK1), regulating caspase 3 activities in apoptosis, Mouse monoclonal to TDT was directly targeted by miR-194-3p. Inhibition of miR-194-3p increased DAPK1 expression and apoptosis in normal HBEpiCs. Importantly, overexpression of miR-194-3p suppressed apoptosis in PM2.5-CSS HBEpiCs. Conclusion These results suggested that miR-194-3p was a protective regulator involved in apoptosis pathway and a potential therapeutic target for treatment of bronchial epithelial injury aggravation induced by PM2.5. and were used as the reference genes, respectively. The sequences of primers for miRNA analysis were as follows (53): U6 from Mir-X miRNA First-Strand Synthesis Kit; hsa-miR-194-3p ATTATTCCAGTGGGGCTGCT. Gene primers for mRNA analysis: forward CTGTGGCATCCACGAAACTA, reverse GTGTTGGCGTACAGGTCTT; forward GTGGATGGTCATTGCAGTTTAAG, reverse TACTGGAGGATGAGAGATGGAG. Luciferase analysis According to the binding site on DAPK1 mRNA 3-untranslated region (3-UTR), a wild-type (wt) gene or a mutated (mut) gene was constructed and cloned into the reporter vector; pMIR-REPORT miRNA expression reporter vector (Obio Technology Corp., Shanghai, China). The HEK293T cells (National Infrastructure of Cell Line Resource, Shanghai, China Infrastructure of Cell Line Resource, China) were transfected with empty vector, DAPK1-3-UTR-wt vector and DAPK1-3-UTR-mut vector with miR-194-3p mimic or scramble control. After 48 h, the transfected cells were analyzed by Dual-Luciferase Reporter Assay System (Promega Corporation, Fitchburg, WI, USA). Immunoblotting analysis Proteins were extracted from cell lysis. The expressions of caspase 3 (anti-pro-caspase 3 from Abcam plc.; anti-cleaved-caspase 3 from Cell Signaling Technology, Inc.) and caspase 9 (anti-caspase 9, from Abcam plc.) in cell lysis were analyzed using 12% SDS-PAGE, whereas DAPK1 (anti-DAPK1, from Sigma-Aldrich Co., St Louis, MO, USA.), AKT (anti-AKT, from Cell Signaling Technology, Inc.) and phosphorylated AKT (anti-phos-AKTser473, from Cell Signaling Technology, Inc.) were analyzed using 10% SDS-PAGE. -Actin (anti–actin, from #TA-09, ZSGB-BIO, China) was the reference control. After being resolved by SDS-PAGE, proteins were transferred to Polyvinylidene Fluoride (PVDF) membranes and then blocked with 5% bovine serum albumin (Sigma-Aldrich Co.) for 1 h. Next, the membranes were separately incubated with primary antibodies at 4C overnight, and then with appropriate HRP-conjugated secondary antibody (from #ZDR-5306 and -5307, ZSGB-BIO) at room temperature for 1 h. After detecting signals using ECL reagents (Merck Millipore, Billerica, MA, USA), gray value of different proteins was quantified with ImageJ v1.28 and normalized to -actin. TUNEL analysis TUNEL (Roche Molecular Systems, Inc., Basel, Switzerland) staining was HIV-1 inhibitor-3 used for measuring DNA fragmentation. HBEpiCs were fixed before detection. The procedures were carried out according to the manufacturers instructions. Caspase-3/7-positive cell detection and TMRM detection The CellEvent? Caspase-3/7 Green detection reagent (Thermo Fisher Scientific) labels nuclei of caspase 3/7-positive cells to report apoptosis. Tetramethylrhodamine, methyl ester reagent (TMRM; Thermo Fisher Scientific) was used to observe the mitochondrial membrane potential. HBEpiCs were loaded with HIV-1 inhibitor-3 50 nM TMRM followed by 20 M CellEvent? detection reagent. Each reagent was incubated for 30 min, and fluorescence signals were observed in living cells. Statistical analysis All experiments were conducted independently at least 3 replicates. Continuous variables are presented as mean SD. Data were analyzed using HIV-1 inhibitor-3 SPSS 13.0 (SPSS Inc., Chicago, IL, USA), and bar graphs were protracted using Prism (version 5.0, GraphPad Software Ltd, San Diego, CA, USA). If the data distribution was normal, comparisons among three or more groups were estimated with an ANOVA test and comparisons between two groups were estimated by two-tailed Students t-test. If the data distribution was not normal, comparisons were estimated by Wilcoxon signed-rank test for nonparametric analysis. p<0.05 was considered statistically significant. Results PM2.5 increased the apoptotic response in cigarette-inflamed HBEpiCs To verify the effect of PM2.5 in cigarette-inflamed bronchial epithelium, HBEpiCs were cultured with normal media, CSS or PM2.5-CSS for 24 h. Typical features of apoptosis were evaluated including mitochondrial membrane potential loss, DNA fragmentation and caspase activation. Caspase activities and mitochondrial membrane potential were detected in living cells (Figure 1A). Compared with normal HBEpiCs, CSS- and PM2.5-CSS-treated HBEpiCs showed a significant loss of mitochondrial membrane potential with an increase of caspase 3/7 activation. Compared with CSS-treated HBEpiCs, PM2.5-CSS cells showed a greater loss of mitochondrial membrane potential with a.