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. 2016 Dec 22;7(12):e2528. doi: 10.1038/cddis.2016.432

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Copyright © 2016 The Author(s)

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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TRIB2 and its promoter activity are regulated by miR-206 and miR-140. (a) TRIB2 was downregulated in miR-206-, miR-140-, or siRNA-treated A549 cells. **P=0.007, miR-140 versus scrambled; **P<0.01, miR-206 or siRNA versus scrambled. Relative values for TRIB2/actin are indicated in the upper panel. (b) Smad3 cDNAs were reduced by siRNA treatment (specific to Smad3) compared with control oligo-treated cultures. Relative values for Smad3/GAPDH cDNAs are indicated in the upper panel. **P<0.01 versus siRNA control. (c) Sketch of TRIB2 promoter luciferase plasmids. WT1, WT2, and WT3 indicate 2.9 kb (at position −2908 to −1), 2.4 kb (at position −2412 to −1), and 1.2 kb (at position −1241 to −1) length promoters of the TRIB2 gene (wild type), respectively. Mut, mutant TRIB2 promoter (The sites at position −2698 and −2692 was mutated). Vec, negative control plasmid. (d) The luciferase levels were higher in 2.9 kb length of the TRIB2 promoter (WT1) plasmid-treated cultures compared with 2.4 kb (WT2), or 1.2 kb (WT3) length promoter-treated cells, especially with TGF-β1 treatment at 24 h. **P<0.01 WT1 with 10 ng/ml TGF-β1 treatment versus untreatment; *P<0.05 WT2 or WT3 versus WT1 with TGF-β1 treatment. (e) SB431542 blocked TGF-β1-mediated enhancement of 2.9 kb length TRIB2 promoter activity. **P<0.01 TGF-β1 treatment versus untreatment, SB431542, or siRNA-Smad3. (f) miR-206 and miR-140 blocked TGF-β1-mediated enhancement of TRIB2 promoter activity. *P<0.05 miR-140 versus TGF-β1 treatment; **P<0.01 miR-206 versus TGF-β1 treatment. (g) Mutant of ‘CAGACA' box at position −2698 and −2692 of the TRIB2 promoter reduced TGF-β1-induced promoter activity. **P<0.01 WT1 with TGF-β1 treatment versus WT1 without TGF-β1, or Mut-promoter. (h) TGF-β1 increased TRIB2 transcription obviously. **P<0.01 TGF-β1 treatment versus untreatment. (i and j) Expression of C/EBP-α or C/EBP-β increased in miR-206- or miR-140-treated A549 cells compared with control treatment. **P<0.01 miR-206, or miR-140, or siRNA versus scrambled oligo control. Relative values for C/EBP-α (or C/EBP-β) versus GAPDH are indicated in the upper panel. Mock, vehicle-treated cells without a reporter plasmid. Scrambled, scrambled oligo control RNA. MiR-206 or miR-140, cells treated with miR-206 or miR-140 oligos. Mu-206 or Mu-140, cells treated with the miR-206 or miR-140 mutation sequence. SiRNA-Smad3 or SiRNA-control, cells treated with small interfering RNA specific to Smad3 or siRNA-control