Pgl4.21 luciferase expression vector

We conducted a promoter methylation assay as previously described (Yang et al., 2016 (link)). In brief, the putative promoter region of the S100A6 gene was polymerase chain reaction (PCR)‐amplified, followed by digestion with the restriction enzyme HindIII and cloning into the pGL4.21 luciferase expression vector (Promega). The vector was then subjected to in vitro methylation using M. SssI, M. Hhal, and M. Hpall methyltransferase enzymes (Invitrogen), which recognize the sequence patterns CG, CGCG, and CCGG, respectively. These three enzymes catalyze in vitro cytosine methylation at the recognized sequence pattern. Finally, a luciferase assay was conducted with the 293T cells using a Dual‐Glo luciferase reporter assay system kit (Promega) 24 h after transfection.

We conducted promoter methylation assay by mimicking a previous study^{23 (link)}. The putative promoter region of the S100A12 gene was PCR amplified, followed by being digested with the restriction enzyme HindIII and being cloned into the pGL4.21 luciferase expression vector (Promega, Madison, WI, USA). The vector was then subjected to in vitro methylation by using the M. SssI methyltransferase enzyme (Invitrogen, Grand Island, NY, USA). M. SssI recognizes the sequence pattern CpG and catalyzes the in vitro cytosine methylation at the recognized sequence pattern. Then, a luciferase assay was conducted in 293 T cell by using a Dual-Glo luciferase reporter assay system kit (Promega, Madison, WI, USA) 24 h after transfection. For the qPCR assays, the sequences of PCR primers are as follow: S100A12: forward primer (5′- CTTACAAAGGAGCTTGCAAAC-3′) and reverse primer (5′- GGTGTGGTAATGGGCAG-3′). 18S: forward primer (5′- GTAACCCGTTGAACCCCATT -3′) and reverse primer (5′- CCATCCAATCGGTAGTAGCG -3′).

The SOX21-AS1 promoter was amplified using the promoter-specific primer pairs (SOX21AS1-promoter-F: ATGAAGCTTCCATGAAGGCGTTCATGGGCCG and SOX21AS1-promoter-R: ATGAAGCTTAGAGGAAGACTCGAGAGGCAGGT). PCR products were digested with the restriction enzyme HindIII and cloned into the pGL4.21 luciferase expression vector (Promega, Madison, WI, USA). Full-length exons 1 and 2 of the SOX21-AS1 PCR products were amplified using individual primer pairs (exon1-F: ATGGAATTCTCTTCTTGGCTCCGGGCAGGGTG and exon1-R: ATGAAGCTTCTGAGCCGGTGCAGAGGGCG; exon2-F: ATGGAATTCGTTTAGGCGAGTGGAGAGTCCG, and exon2-R: ATGCTCGAGAATCTTTAGGACAAAACTGAGC). The PCR products were digested with the restriction enzyme EcoRI and cloned into the pCMV-Tag 2A vector (Stratagene, La Jolla, CA, USA).