Puc18 t vector

The methylation levels of the CpG islands located in the TSS of miR-1224 in Hep3B and HCCLM3 cell lines were assessed using BSP. The forward primer was 5′-GGATTYGGATTAAAATGGT-3′ and the reverse primer was 5′-AATCCTCACCAAAAACAAATA-3′; these were used to amplify the genomic DNA sequences of CpG islands by PCR. The PCR products were separated by 3% agarose gel electrophoresis and then cloned into the pUC18 T-vector (Sangon, Shanghai, China). Following bacterial amplification of the cloned PCR fragments, 10 clones were conducted using DNA sequencing.

Genomic DNA was extracted using a Tissue DNA kit (QIAGEN, Inc., Hilden, Germany) and treated with bisulfate according to the EZ DNA Methylation-Gold™ Kit instruction manual (Zymo Research, USA). The promoter sequence was amplified from the isolated DNA using touchdown PCR, extracted from an agarose gel and loaded into the Puc18-T vector (Sangon Biotech Co., Ltd, Shanghai, China) for TA cloning and sequencing by Shanghai Sangon Biotech Company. The PCR conditions were as follows: 95 °C for 5 min; 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 50 s at 72 °C; and a final extension at 72 °C for 8 min. The BSP primer sequences for CpG region of DAPK1 were as follows: forward, 5′-TTTTTTAAAAAGTAAATAGGTGAGGT-3′ and reverse, 5′-CACCTCCAAAATTCAAATAATTC-3′. The BSP primer sequences for CpG region of p16 were as follows: forward, 5′-TTTGTAGTTAAGGGGGTAGGAGT-3′ and reverse, 5′-CTTTCCTACCTAATCTTCTAAAAAAC-3′. In detail, 10 individual clones from each group were selected and the number of detected methylated CpG sites was divided by the total 10 clones to evaluate the methylation percentage of each CpG site. The total methylation status of the core CpG region within the promoter from each group was calculated by averaging the methylation rate of each CpG site.

Methylation status of the NEAT1 promoter was determined using bisulfite sequencing PCR (BSP). DNA was extracted from DCs using the Ezup Column Animal Genomic DNA Purification Kit (Sangon, Shanghai, China) according to standard procedure. Genomic DNA was modified using sodium bisulfite. The transformed DNA was then amplified via PCR. Primers are listed in Table S2. PCR-amplified products were ligated to pUC18-T vectors (Sangon, Shanghai, China) and transformed into E. coli DH5α cells. IPTG/X-gal plates were used to select qualifying colonies. Ten colonies for each sample were selected and cultured separately in LB^amp+ medium overnight. The obtained plasmids were sequenced by Sangon, Shanghai, China.