Selection of key candidate mRNAs for the validation

To select key candidate mRNAs for the validation stage, we further narrowed down the list of mRNAs that we obtained in TCGA based on a significance level of 1.00 × 10⁻⁴, also integrating the information from the methylation analysis and constructing the PPI network for elucidating biological processes. In detail, we also included those mRNAs whose encoding gene methylation was associated with GC survival, as well as genes in clustered subnetworks within PPI network. A total of 54 mRNAs were included in the validation stage (see Table S3, Supplemental Digital Content 1, http://links.lww.com/CTG/A1).

The validation dataset of GC, including the transcription profile based on {"type":"entrez-geo","attrs":{"text":"GPL6947","term_id":"6947"}}GPL6947 platform (Illumina HumanHT-12 V3.0 expression BeadChip) and clinical information, were obtained from {"type":"entrez-geo","attrs":{"text":"GSE84437","term_id":"84437"}}GSE84437 in GEO (https://www.ncbi.nlm.nih.gov/geo/), followed by background correction and quantile normalization using R package lumi (16). All mRNA expression values were log2-transformed and standardized for comparability with the discovery set as above described.

The association analyses in the validation study were also conducted using Cox regression models, adjusting for age and gender, with one-side P < 0.05 considered as statistically significant.