title: [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].
abstract: 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-α (GRO-α) in cells were determined with real-time fluorescent quantitative reverse transcription-PCR. The protein expression levels of IL-8, MCP-1, and GRO-α 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-α 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-α 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-α 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-α 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.
authors.affiliation: Department of Burns and Plastic Surgery, the 309th Hospital of PLA, Beijing 100091, China.
authors.forename: Wenting
authors.initials: W
authors.lastname: Li
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
authors.forename: Chiyu
authors.initials: C
authors.lastname: Jia
authors.forename: Bin
authors.initials: B
authors.lastname: Yin
authors.forename: Bin
authors.initials: B
authors.lastname: Shu
mesh_terms: D000230:Adenocarcinoma / Q000139:chemically induced*; D000077192:Adenocarcinoma of Lung; D015153:Blotting, Western; D018932:Chemokine CCL2 / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D016209:Interleukin-8; D008168:Lung / Q000187:drug effects* / Q000378:metabolism; D008175:Lung Neoplasms / Q000139:chemically induced*; D035683:MicroRNAs / Q000032:analysis*; D010957:Plasmids; D012333:RNA, Messenger; D012906:Smoke / Q000009:adverse effects*; D012907:Smoking; D014162:Transfection
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D016209:Interleukin-8; D035683:MicroRNAs; D012333:RNA, Messenger; D012906:Smoke