Genomic DNA was isolated using genomic DNA purification kit (Gentra, Minneapolis, MN, USA) and was modified using the CpGenome DNA Modification Kit (Chemicon, Ternecula, Canada) according to the manufacturer's instructions. The primers used for the methylated (M) [(forward) 5'-GATCGTAGGGGATAGTTTCGTGGC-3'; (reverse) 5'-AAAACAACTCTAACACCGCTCCCCG-3'] and unmethylaed (U) [(forward) 5'-TGGATTGTAGGGGATAGTTTTGTGGT-3'; (reverse)5'-CAAAAACAACTCTAACACCACTCCCCA-3'] 20 (link). Real-time quantitative methyaltion-specific PCR (RQ-MSP) reaction contained 0.8 μM of primers, 10 μM of AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Piscataway, NJ, USA), 0.4 μM of ROX Reference Dye 2 (Invitrogen, Carlsbad, CA, USA), and 20 ng of modified DNA was also performed a 7500 Thermo cycler (Applied Biosystems, CA, USA). The reaction condition was 95°C for 5 min, 40 cycles for 10 s at 95°C, 30 s at 62°C (M) or 64°C (U), 72°C for 30 s, and 80°C (M) or 76°C (U) for 30 s, eventually a melting program of one cycle at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, and 60°C for 15 s. The normalized ratio (NM-NKD2) was applied to assess the level of NKD2 promoter methylation in samples. NM-NKD2 was calculated using the following formula: