PMID- 26902276 OWN - NLM STAT- MEDLINE DCOM- 20160426 LR - 20190221 IS - 1009-2587 (Print) IS - 1009-2587 (Linking) VI - 32 IP - 2 DP - 2016 Feb TI - [Effects of microRNA-146a on Fas-associated factor 2 and inflammatory factors in human lung adenocarcinoma A549 cells under the stimulation of cigarette smoke extract]. PG - 97-104 LID - 10.3760/cma.j.issn.1009-2587.2016.02.008 [doi] AB - OBJECTIVE: Under the premise of smoke inhalation injury, to explore the effects of microRNA-146a on Fas-associated factor 2 (FAF-2) and inflammatory factors in human lung adenocarcinoma A549 cells under the stimulation of cigarette smoke extract (CSE). METHODS: (1) The pMIR-FAF-2 recombinant plasmid and the pMIR-FAF-2 recombinant mutated plasmid were constructed. Human embryonic kidney 293 (HEK-293) cells of the third passage were divided into 3 groups according to the random number table, with 5 wells in each group. Cells in plasmid+ microRNA control group were transfected with pMIR-FAF-2 recombinant plasmid, pRL-TK plasmid, and microRNA control; cells in plasmid+ microRNA-146a group were transfected with pMIR-FAF-2 recombinant plasmid, pRL-TK plasmid, and microRNA-146a mimics; cells in mutated plasmid+ microRNA-146a group were transfected with pMIR-FAF-2 recombinant mutated plasmid, pRL-TK plasmid, and microRNA-146a inhibitor. After culture for 24 h, the relative luciferase activity in cells was assessed by dual-luciferase reporter gene assay. (2) Human lung adenocarcinoma A549 cells of the third passage were divided into 3 groups according to the random number table, with 4 wells in each group. Cells in microRNA control group were transfected with microRNA control; cells in microRNA-146a enhancement group were transfected with microRNA-146a mimics; cells in microRNA-146a inhibition group were transfected with microRNA-146a inhibitor. After culture for 24 h, the mRNA expression levels of microRNA-146a and FAF-2 in cells were determined with real-time fluorescent quantitative reverse transcription-PCR. (3) A549 cells of the third passage were stimulated by 0.8% CSE for 24 h after being divided and treated with the same method used in experiment (2). The mRNA expression levels of FAF-2, IL-8, monocyte chemotactic protein-1 (MCP-1), and growth-regulated oncogene-alpha (GRO-alpha) in cells were determined with real-time fluorescent quantitative reverse transcription-PCR. The protein expression levels of IL-8, MCP-1, and GRO-alpha in A549 cell culture supernatant were determined by enzyme-linked immunosorbent assay. The protein expression level of cyclooxygenase 2 (COX-2) of cells was assessed by Western blotting. Data were processed with one-way analysis of variance and LSD test. RESULTS: (1) The pMIR-FAF-2 recombinant plasmid and pMIR-FAF-2 recombinant mutated plasmid were confirmed with successful construction. The relative luciferase activity in HEK-23 cells of plasmid+ microRNA control group was close to that of mutated plasmid+ microRNA-146a group (P>0.05). The relative luciferase activity in HEK-23 cells of plasmid+ microRNA-146a group was significantly lower than that of plasmid+ microRNA control group and mutated plasmid+ microRNA-146a group (with P values below 0.01). (2) The expression level of microRNA-146a in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P>0.05), and they were both significantly lower than the expression level of microRNA-146a in A549 cells of microRNA-146a enhancement group (with P values below 0.01). The mRNA expression level of FAF-2 in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P>0.05), and they were both significantly higher than the mRNA expression level of FAF-2 in A549 cells of microRNA-146a enhancement group (with P values below 0.05). (3) After stimulation of CSE, the mRNA expression level of FAF-2 in A549 cells of microRNA control group (1.46+/-0.21) was close to that of microRNA-146a inhibition group (1.43+/-0.34, P>0.05), which were both significantly higher than the mRNA expression level of FAF-2 in A549 cells of microRNA-146a enhancement group (0.57+/-0.11, with P values below 0.05). The mRNA expression levels of IL-8, MCP-1, and GRO-alpha in A549 cells of microRNA-146a enhancement group were significantly lower than those of microRNA control group and microRNA-146a inhibition group (with P values below 0.01). The mRNA expression levels of IL-8, MCP-1, and GRO-alpha in A549 cells of microRNA-146a inhibition group were significantly higher than those of microRNA control group (with P values below 0.05). The protein expression levels of IL-8, MCP-1, and GRO-alpha in A549 cell culture supernatant of microRNA-146a enhancement group were significantly lower than those of microRNA control group and microRNA-146a inhibition group (with P values below 0.05). The protein expression level of IL-8 in A549 cell culture supernatant of microRNA-146a inhibition group was close to that of microRNA control group (P>0.05), while the protein expression levels of MCP-1 and GRO-alpha in A549 cell culture supernatant of microRNA-146a inhibition group were significantly lower than those of microRNA control group (with P values below 0.05). The protein expression level of COX-2 in A549 cells of microRNA-146a enhancement group was significantly lower than the levels of microRNA control group and microRNA-146a inhibition group (with P values below 0.05). The protein expression level of COX-2 in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P>0.05). CONCLUSIONS: In A549 cells, after being transfected with microRNA-146a and stimulated by CSE, microRNA-146a can decrease the expression of FAF-2 through integrating with the 3'-untranslated region of target gene FAF-2, thereby decrease the expression of inflammatory factors. FAU - Li, Wenting AU - Li W AD - Department of Burns and Plastic Surgery, the 309th Hospital of PLA, Beijing 100091, China. FAU - Liu, Zhen AU - Liu Z FAU - Jia, Chiyu AU - Jia C FAU - Yin, Bin AU - Yin B FAU - Shu, Bin AU - Shu B LA - chi PT - Journal Article PT - Randomized Controlled Trial PT - Research Support, Non-U.S. Gov't PL - China TA - Zhonghua Shao Shang Za Zhi JT - Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns JID - 100959418 RN - 0 (CCL2 protein, human) RN - 0 (Chemokine CCL2) RN - 0 (Interleukin-8) RN - 0 (MicroRNAs) RN - 0 (RNA, Messenger) RN - 0 (Smoke) SB - IM MH - Adenocarcinoma/*chemically induced MH - Adenocarcinoma of Lung MH - Blotting, Western MH - Chemokine CCL2/*metabolism MH - Enzyme-Linked Immunosorbent Assay MH - HEK293 Cells MH - Humans MH - Interleukin-8 MH - Lung/*drug effects/metabolism MH - Lung Neoplasms/*chemically induced MH - MicroRNAs/*analysis MH - Plasmids MH - RNA, Messenger MH - Smoke/*adverse effects MH - Smoking MH - Transfection EDAT- 2016/02/24 06:00 MHDA- 2016/04/27 06:00 CRDT- 2016/02/24 06:00 PHST- 2016/02/24 06:00 [entrez] PHST- 2016/02/24 06:00 [pubmed] PHST- 2016/04/27 06:00 [medline] AID - 10.3760/cma.j.issn.1009-2587.2016.02.008 [doi] PST - ppublish SO - Zhonghua Shao Shang Za Zhi. 2016 Feb;32(2):97-104. doi: 10.3760/cma.j.issn.1009-2587.2016.02.008.