Journal: Cell Death & Disease
Article Title: Reactive oxygen species-responsive miR-210 regulates proliferation and migration of adipose-derived stem cells via PTPN2
Figure Lengend Snippet: Signal pathway and transcription factor involved in miR-210 upregulation. ROS generators induce miR-210 level not only by HIF-1 α stabilization but also by phosphorylation of PDGFR- β , Akt and ERK1/2. In addition, NF- κ B and Elk1 are involved in the transcription of miR-210. ( a ) Although hypoxia (Hyp.) (2%) induced HIF-1 α level, low concentrations of antimycin (Ama.) and rotenone (Rot.) did not in western blot analysis. ( b ) HIF-1 α was not reduced by ROS scavenger, NAC. ( c ) Hypoxia-induced miR-210 expression was not reduced by an HIF-1 α inhibitor (Yc-1, 50 and 100 μ M). ( d ) Phosphorylation of PDGFR- β , Akt and ERK 1/2 molecules was significantly increased by antimycin (10 nM) and rotenone (10 pM) treatment according to western blot analysis. ( e , f ) Antimycin- ( e ) and rotenone- ( f ) induced miR-210 expressions were significantly reduced by AG1296 (a PDGFR inhibitor, 10 μ M), LY-294002 (a PI3K/Akt inhibitor, 5 μ M) and U0126 (an MAPK/ERK inhibitor, 5 μ M). ( g ) The promoter region upstream of miR-210 was analyzed using Patch 1.0 and AliBaba 2.1 program, and NF- κ B and Elk1 were found to be candidates for miR-210 transcription factors. ( h , i ) Phosphorylation of NF- κ B and Elk1 was induced by antimycin (10 nM, h ) and rotenone (10 pM, i ) treatment. ( j , k ) In addition, antimycin- ( j ) and rotenone- ( k ) induced miR-210 expression was significantly attenuated by transfection of siRNA for NF- κ B (20 nM) and Elk1 (20 nM). ( l ) ChIP assay shows that antimycin and rotenone treatment significantly increased the signal intensity of PCR products, which indicated that phosphorylated NF- κ B and Elk1 could bind to the upstream region of miR-210 promoter. ** P
Article Snippet: For the inhibition study, 0.1 mM NAC (ROS scavenger; Sigma-Aldrich, St. Louis, MO, USA), 50 and 100 μ M Yc-1 (an HIF-1α inhibitor; Sigma), 5 μ M LY294002 (PI3K/Akt inhibitor; Calbiochem, San Diego, CA, USA), 5 μ M U0126 (MAPK/ERK inhibitor; Calbiochem), 10 μ M AG1296 (PDGFR inhibitor; Calbiochem) and 0.001–1 μ M ethyl-3,4-dephostatin (PTPN2 inhibitor; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.
Techniques: Western Blot, Expressing, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction