S in human get Zebularine colorectal cancer. Methylation of DIRAS1 may promote colorectal carcinogenesis and progression. Additional filesAdditional file 1: Table S1. Nucleotide sequences of the primers used in this study. (XLS 25 kb) Additional file 2: Figure S1. Methylation status and expression of DIRAS1 in primary colorectal cancer and adjacent samples from TCGA. (A) The correlation of methylation of each CpG site (HM450) and expression of DIRAS1. (B) The methylation status of the CpG site (cg05228284, HM450) is correlated to loss of/reduced DIRAS1 expression in 217 cases of colorectal cancer samples and 16 cases of adjacent samples. (R = -0.278, P < 0.0001). (C) The methylation status of the CpG site (cg05228284, HM450) is correlated to loss of/reduced DIRAS1 expression in 234 cases of colorectal cancer. (P < 0.0001). (D) Kaplan-Meier curves show the association of overall survival rate of colorectal cancer patients with the methylation status of CpG site (cg05228284, HM450). (P > 0.05). (E) Kaplan-Meier curves show the association of overall survival rate of colorectal cancer patients with the expression levels of DIRAS1. (P > 0.05). (TIF 572 kb)Discussion The Ras superfamily is divided into five subfamilies according to their sequence homology and biochemical properties. The five families are Ras, Rab, Rho, Ran, and Arf. Ras members may activate downstream signaling by binding to GTP or serve as an inactive form by binding to GDP. When bound to GTP, Ras proteins associate with effectors, resulting in the propagation of downstream signaling. Most members of this superfamily have been widely studied and identified as oncoproteins [17, 18]. DIRAS1 may serve as a competitive inhibitor of Ras and antagonize Ras-mediated ERK1/2 signaling to promote cell apoptosis and suppress cell invasion. The expression levels of DIRAS1 were found to be reduced in human breast cancer and esophageal cancer [13, 19]. In this study, we found that DIRAS1 is frequently methylated in human CRC, and the expression of DIRAS1 is regulated by promoter region methylation. In TCGA database, the promoter region methylation was inversely associated with DIRAS1 expression in colorectal cancer. The levels of DNA methylation were significantly higher in colorectal cancer samples compared to adjacent tissue samples according to HMK450 methylation array data in human colorectal cancer. These data further supported our results. Methylation PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 of DIRAS1 was associated with TNM stage and short survival. These results suggest that methylation of DIRAS1 may serve as a marker of poor prognosis in human colorectal cancer. In our studied cohort, patients were received different chemotherapeutic regimens. It limits to further analyze the association of DIRASAbbreviations BSSQ: Bisulfite sequencing; CRC: Colorectal cancer; DAC: 5-Aza-2-deoxycytidine; DKO: DNMT1 and DNMT3b double knockout; ECM: Extracellular matrix; GAPDH: Glyceraldehyde-3-phosphatedehydrogenas; IHC: Immunohistochemistry; M: Methylated; MSP: Methylation-specific PCR; Rig: Ras-related inhibitor of cell growth; SD: Standard deviation; STS: Staurosporine; TSS: Transcriptional start sites; U: Unmethylated Acknowledgements We sincerely thank Xiaomo Su for preparing experiments. Funding This work was supported by grants from the National Basic Research Program of China (973 Program No. 2012CB934002), National Key Research and Development Programme of China (2016YFC1303600); National Key Scientific Instrument Special Programme of China (.
