And extracted by phenol/chloroform. Bisulfite treatment was performed as previously described [14]. Methylation-specific PCR (MSP) primers were designed according to genomic sequences around transcriptional start sites (TSS) and synthesized to detect unmethylated (U) and methylated (M) alleles. Bisulfite sequencing (BSSQ) was performed as previously described [15]. BSSQ products were amplified by primers flanking the targeted regions including MSP products. Sequences of the MSP primers and bisulfite sequencing primers are shown in Additional file 1: Table S1. To obtain more evidence supporting our discovery, the expression and the methylation status of DIRAS1 in The Cancer Genome Atlas database were analyzed in primary colorectal cancer and adjacent tissue samples (Additional file 2: Figure S1).ImmunohistochemistryImmunohistochemistry (IHC) was performed in primary colorectal cancer samples and paired adjacent tissue samples. The DIRAS1 antibody was diluted 1:100 (Bioworld Technology, Beijing, China). The staining PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 intensity and extent of the staining area were scored using the German Luteolin 7-glucoside web Semi-quantitative scoring system as previously described [15].Plasmid construction and transfectionColorectal cancer cell lines were split to a low density (30 confluence) 12 h before treatment. Cells were treated with 5-aza-2-deoxycytidine (DAC, Sigma, St. Louis, MO) at a concentration of 2 M. Growth medium conditioned with DAC at a concentration of 2 M was exchanged every 24 h for a total of 96 h of treatment.RNA isolation and semi-quantitative reverse transcription PCRTotal RNA was isolated by Trizol reagent (Life Technologies, MD, USA). First-strand cDNA was synthesized according to the manufacturer’s instructions (Invitrogen, CA, USA). The primer sets for DIRAS1 were designed to span intronic sequences between adjacent exons to control for genomic DNA contamination. Semi-quantitative reverse transcription PCR (RT-PCR) was amplified for 34 cycles. Glyceraldehyde-3-phosphatedehydrogenas (GAPDH) was used as an internal control. Primer sequences are shown in Additional file 1: Table S1.Human full-length DIRAS1 coding sequences (CDS) was amplified and subcloned as described previously [4]. The primers used were 5-CGCGGATCCATGCCGGAACA GAGTAACG-3 (F) and 5-CCGCTCGAGTCACATGA GGGTGCATTTGC-3 (R). DIRAS1 expressing lentiviral or empty vectors were packaged using the ViraPowerTM lentiviral expression system (Invitrogen, San Diego, CA, USA). Lentivirus was added to the growing medium of DLD1 and RKO cells, and DIRAS1 stably expressed cells (DLD1-DIRAS1 and RKO-DIRAS1 cells) and control cells (DLD1-Vector and RKO-Vector cells) were selected by blasticidin (Invitrogen, San Diego, CA, USA) at a concentration of 5 g/ml. The selected siRNAs targeting DIRAS1 sequences were as follows: sense: 5-CCACCAGGCAA UAACCACATT-3; antisense: 5-UCGAUGUUGAGG CUCAUGUTT-3; negative control sequences were as follows: sense: 5-UUCUCCGAACGUGUCACGUTT-3; antisense: 5-ACGUGACACGUUCGGAGAATT-3.Western blotProtein preparation and Western blot were performed as described previously [16]. The antibodies for WesternZheng et al. Clinical Epigenetics (2017) 9:Page 3 ofblot analysis were as follows: rabbit anti-DIRAS1 (Bioworld Technology, Beijing, China), rabbit anti-MMP2 (Bioworld Technology, Beijing, China), rabbit antiMMP9 (Bioworld Technology, Beijing, China), and rabbit anti-cleaved caspase 3 (Bioworld Technology, Beijing, China). Rabbit anti-GAPDH (Bioworld Technology, Beijing, China) was use.