Figure 2. Down-regulation of miR-34b was related with DNA upstream of CpG site methylation and mediated by DNMT3a that was involved in regulating VSMCs calcification. (A) A schematic illustration of the location of the CpG islands upstream of miR-34b DNA. (B) BSP showed that the methylation rate of CpG sites of miR-34b DNA was significantly higher in VSMCs with 3.5 mM of Pi treatment than that of control. Meanwhile, 5-aza (10 μmol/L), a DNA methyltransferase inhibitor, decreased the methylation level of CpG sites of miR-34b in VSMCs. (C) The expression of miR-34b was detected by qRT-PCR in VSMCs treated with 3.5 mM of Pi or 3.5 mM of Pi + 10 μmol/L of 5-aza. (D) qRT-PCR showed the different expression levels of DNMT1, DNMT3a and DNMT3b in VSMCs cultured in 3.5 mM of Pi or 3.5 mM Pi + 10 μmol/L of 5-aza. (E) Western blot analysis showed that the different levels of DNMT3a protein in VSMCs treated with 3.5 mM of Pi or 3.5 mM Pi + 10 μmol/L of 5-aza. (F) qRT-PCR detected the expression of miR-34b after knocking down DNMT3a with DNMT3a siRNA in VSMCs. (G–I) ALP activity, OC secretion and Runx2 expression were determined in VSMCs induced by 3.5 mM of Pi after transfecting with scramble siRNA or DNMT3a siRNA. (J) Cultured VSMCs were subjected to ChIP using the anti-DNMT3a antibody, followed by qPCR analysis using primers annealing to the promoter sequence of miR-34b. n = 3. The data were expressed as mean ± SD, *p < 0.05; **p < 0.005; ***p < 0.0005.