Overexpression of miR-29a/b/c suppresses DNMT3A expression in directly manner.

<p>(A) Schematic diagram of predicted miR-29a/b/c target site in the 3’ UTR of DNMT3A. (B) MiR-29a/b/c regulate DNMT3A by directly binding to its 3’ UTR. 293T cells in 24-well plates were transfected with 3’ UTR reporter (50 ng), renilla vector (pRL-TK, 20 ng), and miRNA mimics (100 nM). After 24 h transfection, the cells were collected for dual-luciferase assay. (C) Anti-miR-29a/b/c elevates the luciferase activity of pGL3-DNMT3A. 3T3-L1 cells in 24-well plates were transfected with 3’ UTR reporter (50 ng), renilla vector (pRL-TK, 20 ng), and miRNA antisense (100 nM). After 48 h transfection, the cells were collected for dual-luciferase assay. * <i>p</i> < 0.05, ** <i>p</i> < 0.01. (D) The overexpression or inhibition efficiency of miR-29a/b/c mimics (left) or antisense (right) in 3T3-L1 cells. 100 nM miR-29a/b/c mimics or antisense was transfected in 3T3-L1 cells, the expression of miR-29a/b/c was detected by real-time PCR after 48 h transfection. * <i>p</i> < 0.05, ** <i>p</i> < 0.01. (E)—(F) MiR-29a/b/c regulate DNMT3A protein level. 80% density 3T3-L1 cells were transfected with 100 nM miR-NC, miR-29a, miR-29b, miR-29c mimics (E) or antisense (F) for 48 h. The expression of DNMT3A was analyzed by western blotting. Tubulin was used as a loading control. The data were presented as means ± SEM. ** <i>p</i> < 0.01. (G) 3T3-L1 cells were transfected with 100 nM miR-NC, miR-29a, miR-29b or miR-29c mimics in CI 0h stage, the cells were collected in CI 24h or CI 48h stage for western blotting analysis. The data were presented as means ± SEM. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.