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. 2011 Jul 11;121(8):3343–3356. doi: 10.1172/JCI44957

Figure 8. p53 activation upregulates CTGF synthesis via repression of the miR-17-92 cluster gene.

Figure 8

(A) HepG2 cells (1.0 × 105) were cotransfected with pTS-589 and pRL-TK for 48 hours and treated with nutlin-3a (20 μM) or recombinant TGF-β (10 ng/ml) for 24 hours. Firefly luciferase and Renilla luciferase activity was measured and is presented as relative luminescence values for firefly luciferase versus Renilla luciferase (F/R). n = 4/group. (B and C) HepG2 cells (1.0 × 105) were treated with nutlin-3a (20 μM) or vehicle for 24 hours. (C) Real-time RT-PCR analysis of miR-17-92 mRNA (B), MIR18A, MIR19A, and MIR19B miRNA expression; n = 3/group. Statistical analyses were performed by the paired t test (A–C). (D) HepG2 cells were transfected with a mixture of antisense of MIR18A, MIR19A, and MIR19B at 100 nM each or negative control at 300 nM for 2 days. Expression of CTGF protein was assessed by Western blotting. (E) HepG2 cells were transfected with a mixture of precursor of MIR18A, MIR19A, and MIR19B at 10 nM each or negative control at 30 nM for 2 days and cultured with nutlin-3a (20 μM) or vehicle for 24 hours. Expression of CTGF protein was assessed by Western blotting. (F) Expression of miR-17-92 mRNA in isolated hepatocytes was assessed by real-time RT-PCR. Cre(+), Mdm2fl/flalb-cre; cre(–), Mdm2fl/fl; 5 mice per group.