Figure 3

miR-584-3p interacts with AGO2 to repress the YY1-facilitated MMP-14 transcription. (a) Real-time quantitative RT-PCR assay showing the expression of miR-584-3p in normal gastric epithelial GES-1 cells and gastric cancer cell lines SGC-7901, AGS, MKN-45, and MKN-28 (mean ± SD, n = 4). (b and c) Real-time quantitative RT-PCR assays indicating the expression of miR-584-3p in gastric cancer cells transfected with empty vector (mock), miR-584-3p precursor, negative control inhibitor (anti-NC, 100 nmol/L), or anti-miR-584-3p inhibitor (100 nmol/L) (mean ± SD, n = 4). (d and e) Western blot assay showing the expression of MMP-14 and VEGF₁₆₅ in gastric cancer cells transfected with mock, miR-584-3p precursor, anti-NC (100 nmol/L), or anti-miR-584-3p inhibitor (100 nmol/L). (f and g) Dual-luciferase assay indicating the MMP-14 promoter activity (normalized to pGL3-Basic) in gastric cancer cells transfected with mock, miR-584-3p precursor, anti-NC (100 nmol/L), or anti-miR-584-3p inhibitor (100 nmol/L; mean ± SD, n = 4). (h and i) Western blot and dual-luciferase assays showing the expression of AGO2 and MMP-14 and activity of MMP-14 promoter reporter [pGL3-MMP14 (−1246/ + 199)] in gastric cancer cells stably transfected with mock or miR-584-3p precursor, and those co-transfected with scramble shRNA (sh-Scb) or AGO2 shRNA (sh-AGO2). (j) ChIP (using AGO2 antibody) and qPCR assay indicating the binding of AGO2 (normalized to 20% input DNA) to MMP-14 promoter in gastric cancer cells treated with RNase H or RNase A (mean ± SD, n = 4). (k) co-IP and western blot assays showing the interaction of AGO2 with EZH2, EHMT2, and YY1 in MKN-45 cells stably transfected with mock or miR-584-3p precursor, running under the same experimental conditions (full-length blots are presented in Supplementary Figure S7). (l) ChIP and qPCR assay indicating the enrichment (normalized to mock + sh-Scb or mock + mock) of AGO2, EZH2, EHMT2, H3K27me3, H3K9me2, and YY1 on MMP-14 promoter in MKN-45 and AGS cells stably transfected with mock or miR-584-3p precursor, and those co-transfected with sh-Scb, sh-AGO2, mock, or YY1 (mean ± SD, n = 5). *P < 0.01 vs. GES-1, mock, anti-NC, pGL3-Basic, mock + sh-Scb, mock + mock, or IgG. **P < 0.01 vs. mock or anti-NC.