Total DNA was prepared by the proteinase K method. MSP and BS were performed as previously described [27 (link), 28 (link)]. The in vitro methylation DNA (IVD) serving as a positive control was the genomic DNA from HepG2 cells treated in vitro with Sss I methyltransferase (New England Biolabs, MA, USA), and the negative control was the DNA from normal human peripheral lymphocytes as described. MSP products were analyzed using a 2% agarose gel electrophoresis. Bisulfite-treated sample DNA was amplified using BS primers, and the PCR products were gel purified and ligated into pEASY-T1 vector (TransGen Biotech, Beijing, China). Colonies were randomly selected for plasmid isolation using Wizard miniprep kits (Promega, Shanghai, China) and were subjected to sequence with the M13 reverse primer via automated sequencing (BGI Sequencing, Beijing, China) as described [27 (link)].