Next Generation Sequencing Facilitates Quantitative Analysis of Cystine-regulated Transcriptomes

Purpose: To identify differentially expressed genes regulated by cystine. Methods: HCT116 or RKO cells were cultured for 48 hours in cystine-free or 25μM cystine-containing conditional media, and three independent samples were collected by Trizol reagent and subjected to mRNA-sequencing by the LC Bio (Zhejiang, China) and data analysis Results: we identified cystine upregulated 145 genes and downregulated 169 genes in both HCT116 and RKO cells. GO enrichment analysis revealed that cystine regulated biological functions of G1/S transition of mitotic cell cycle and regulation of DNA replication biosynthetic process.In addition, KEGG enrichment analysis showed that mammalian target of rapamycin (mTOR) signaling pathway, a well-known nutrient sensor, was regulated by cystine. Furthermore,we noticed that SESN2 was one of the most significantly downregulated genes by cystine in both cell lines, and confirmed that cystine activates mTORC1 via the GCN2-ATF4-SESN2 axis in colon cancer cells. Conclusions: Our study established that cystine activates mTORC1 through GCN2-ATF4-SESN2 axis. The mRNA profiles of HCT116 and RKO cells cultured in cystine-free or 25μM cystine-contained media were generated for High-throughput sequencing, in triplicate, and analyzed the cystine-regulated transcriptomes