Depletion of Sirtuin 1 (SIRT1) Leads to Epigenetic Modifications of Telomerase (TERT) Gene in Hepatocellular Carcinoma Cells
Figure 2
Bisulfite sequencing of TERT gene in different HCC cell lines following SIRT1 depletion.
Bisulfite-modified genomic DNAs of HCC cell lines expressing the indicated shRNA was amplified by PCR using two independent primer sets (BSP-1 and BSP-2) spanning the proximal promoter region of TERT. PCR products were cloned into pCR2.1-TA vectors, and at least 8 clones were sequenced and analyzed for their methylation status by using the QUMA online tools (http://quma.cdb.riken.jp/). The percentage of methylated clones at each CpG site is depicted by a pie chart. The number of the pie charts corresponds to the number of the CpG positions on the map. In SK-HEP-1 and SNU423 cells, SIRT1-depletion enhanced CpG methylation between −266 to −240 bp upstream of TERT transcription start site. In contrast, SIRT1-depletion reduced CpG methylation between +74 to +122 bp in SK-HEP-1 cells and between +82 to +122 bp in Hep3B cells. * P<0.05, ** P<0.01 versus lentiviral control shRNA-transduced cells by the Mann-Whitney U-test.