Figure 8

Modulation of DNMT-regulating miRNAs affects the expression of INK4/p16 and consequent cell growth control. Treatment with 5-Aza in (a) empty vector-transfected and (b) Tat-transfected cells. Treatment with 5-Aza induces the expression of miRNAs containing CpG islands in Tat-transfected cells, indicating that down-regulation of these miRNAs depends on hypermethylation. (c) Treatment with 5-Aza was also able to restore the expression of INK4/p16 in Tat-transfected cells, thus suggesting that silencing of INK4/p16 is due to hypermethylation. (d) Ectopic modulation of DNMT-regulating miRNAs was achieved using synthetic mimic or inhibitors. Relative expression of INK4/p16 was then checked by RT-qPCR. A decreased expression of the gene was observed following inhibition of miRNAs, which leads to up-regulation of DNMTs and consequent hypermethylation of target genes. Conversely, increased expression of endogenous miRNAs, which leads to downregulation of DNMTs, results in over-expression of INK4/p16 (p < 0.05). NC: Mimic negative control; M: Either hsa-miR152 or hsa-miR29 mimic; NCI: Inhibitor negative control; I: Either hsa-miR152 or hsa-miR29 inhibitor. (e-f) Cell proliferation analysis in cells transfected with miRNA mimics/inhibitors for DNMT1 (e) and DNMT3a/b (f). Inhibition of the endogenous miRNAs enhances cell growth.