Figure 6
Phe increased global DNA methylation, expression of DNMT1, DNMT3a and DNMT3b and the methylation level of the 5 CpG sites located at the putative transcription start sites of the miR-133a loci in H9C2 cells.
(A) Phe increased global DNA methylation in a dose dependent manner. Means of exposures not sharing a common letter are significantly different at P < 0.05 (Dunnett). (B,C) Based on the results of Western blotting, we found that various doses of Phe were capable of increasing the expression of DNMT1, DNMT3a and DNMT3b in H9C2 cells. Intensities of protein bands were quantified using densitometry. Results are expressed as multiples (×folds) of optical density of target protein and the alpha-tubulin determined in the control. The mean protein expression from the control was designated as 1 in the graph. Values (means ± S.D.) are representative of data obtained in three independent experiments (n = 3). Treatments not sharing a common letter are significantly different at P < 0.05 as assessed by one-way ANOVA followed by the Dunnett’s test. (D) Schematic representation of the locations of the 5 CpG sites closely located at the putative transcription start sites of the miR-133a loci (−1000–−1300 bp) identified by the UCSC and MethPrimer database. Red lines indicate the positions of the CpG sites. (E) From the results of HRM assay, the melting curves of the control overlapped with the melting curves of the not methylated standard sample (blue). In Phe treated groups, variation in methylation was evident between not methylated and 100% methylated (red) standard sample. The position of the melting curves indicated that Phe exposure increased the methylation level of the 5 CpG sites located at the putative transcription start sites of the miR-133a loci.
