www.aging-us.com AGING 2019, Vol. 11, No. 19 Research Paper Association of a novel seven-gene expression signature with the disease prognosis in colon cancer patients Haojie Yang1,*, Hua Liu1,*, Hong-Cheng Lin2,*, Dan Gan1, Wei Jin1, Can Cui1, Yixin Yan3, Yiming Qian3, Changpeng Han1, Zhenyi Wang1 1Department of Coloproctology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China 2Department of Coloproctology, The Sixth Affiliated Hospital of Sun Yat-sen University, Gastrointestinal and Anal Hospital of Sun Yat-sen University, Guangzhou 510655, China 3Department of Emergency Medicine, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China *Equal contribution Correspondence to: Changpeng Han, Zhenyi Wang; email: [email protected], [email protected] Keywords: colon cancer, gene expression profile, signature, ceRNA Received: July 12, 2019 Accepted: October 7, 2019 Published: October 15, 2019 Copyright: Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Older patients who are diagnosed with colon cancer face unique challenges, specifically regarding to cancer treatment. The aim of this study was to identify prognostic signatures to predicting prognosis in colon cancer patients through a detailed transcriptomic analysis. RNA-seq expression profile, miRNA expression profile, and clinical phenotype information of all the samples of TCGA colon adenocarcinoma were downloaded and differentially expressed mRNAs (DEMs), differentially expressed lncRNAs (DELs) and differentially expressed miRNAs (DEMis) were identified. A competing endogenous RNA (ceRNA) network was constructed further and DEMs related with prognosis in the ceRNA network was screened using Cox regression analysis. Risk score models for predicting the prognosis of colon cancer patients were built using these DEMs. A total of 1476 DEMs, 9 DELs, and 243 DEMis between the tumor and normal samples were identified and functional enrichment analyses showed that the DEMs were significantly enriched in the nervous system development, ribosome biogenesis pathways in eukaryotes, and drug metabolism cytochrome P450. Twelve DEMs related with prognosis were screened from the ceRNA network. Thereafter, the risk score models of prognostic DEMs were obtained, involving seven DEMs (SGCG, CLDN23, SLC4A4, CCDC78, SLC17A7, OTOP3, and SMPDL3A). Additionally, cancer stage was identified as a prognostic clinical factor. This prognostic signature was further validated in two independent datasets. Our study developed a seven-mRNA and one-clinical factor signature that are associated with prognosis in colon cancer patients, which may serve as possible biomarkers and therapeutic targets in the future. INTRODUCTION significant progress in tailored therapy, the high migration and invasion capabilities of this tumor have been a Colon cancer is the third most common cancer, with bottleneck in reducing the mortality, keeping its 5-year increasing morbidity and mortality worldwide [1]. The survival rate under 12% [2, 3]. It has been reported that morbidity of colon cancer increases along with the the prognosis of colon cancer mainly depends on the increase of age. In fact, more than 60% of people who clinicopathological features and the tumor stage. developed cancer are order than 65. Despite the Nevertheless, due to the remarkable disease heterogeneity, www.aging-us.com 8710 AGING it is difficult to determine the prognosis of patients based downregulated) DELs, and 243 (113 upregulated and on these traditional factors [4]. Therefore, a better 130 downregulated) DEMis were identified. Top10 understanding of the pathogenesis and identification of genes (in ascending order according to the adjusted P new promising prognostic biomarkers are essential for value) were selected from DEMs, DELs, and DEMis to development of effective therapies for colon cancer draw the heatmaps, as shown in Figure 1. patients. GO enrichment analysis of DEMs A recent study has reported that the identification of prognostic signature based on the genomic or With a threshold of adjusted P value < 0.01, 13 BPs, transcriptome data can improve our understanding of 14 CCs, and 5 MFs were significantly enriched by the cancer progression and survival rate [5]. Presently, DEMs. The top 5 terms for the three categories are many prognostic gene-expression signatures have been shown in Figure 2A. The seven most significant terms identified in various human cancers which help in are shown in detail. According to the genes in term, predicting the cancer prognosis outcomes [6–8]. the clustering of genes was carried out to construct the Among the various gene-expression signatures in phylogenetic tree (Figure 2B). In terms of the colon cancer, the six-cluster gene expression ColoPrint involved BP terms, the DEMs were significantly [9] and Colon OncotypeDx [7] have been widely enriched in chemical synaptic transmission, axon verified in retrospective studies. Recently, a study has guidance, nervous system development, cell adhesion, reported that the protein-coding genes make up less and transport. From the perspective of CC terms, the than 2% of the whole genome sequences, and the encoding proteins were mainly located in an integral noncoding genes are transcribed into noncoding component of the plasma membrane, plasma RNAs, such as microRNAs (miRNAs) and long membrane, and apical plasma membrane. noncoding RNAs (lncRNAs) [10]. Some of the recent studies have demonstrated the competing endogenous Pathway enrichment analysis RNA (ceRNA) activity of lncRNAs, as natural miRNA decoys in human pathophysiological conditions, According to the KEGG pathway enrichment analysis including cancer [11]. Therefore, systematic analysis of the DEMs obtained above by GSEA, a total of 67 of lncRNA-associated ceRNA network may help to significantly enriched pathways were identified explore and identify more prognostic gene-expression (adjusted P value < 0.05), among which 16 were signatures in colon cancer. upregulated pathways (normalized enrichment score (NES) > 0) and 51 were downregulated pathways In this study, we aimed to screen newer prognostic (NES < 0). KEGG ribosome biogenesis in eukaryotes signatures for colon cancer through the analysis of had the largest NES in the significantly upregulated RNA-seq expression profile and miRNA expression pathways (Figure 2C). In addition, DNA replication, profiles. Based on the differentially expressed mRNAs RNA degradation, IL17 signaling pathway, p53 (DEMs), lncRNA (DELs), and miRNA (DEMis) data signaling pathway, and oxytocin signaling pathway obtained between the tumor and normal samples, a were also significantly upregulated. Among the ceRNA network was constructed and DEMs involved in significantly downregulated pathways, drug this network were selected for the construction of metabolism cytochrome P450 had the largest NES prognostic risk model. Our study presented some novel (Figure 2D). Additionally, chemical carcinogenesis biomarkers with a potential prognostic value, and and mineral absorption pathways were also provided a preliminary bioinformatic evidence for downregulated. understanding the complex mechanism of colon cancer progression. Co-expression analysis RESULTS For the screened DELs and DEMis, the correlation coefficients and statistical significance degree between Differential analysis them and DEMs were calculated, respectively, and the gene pairs with |r| > 0.6 and an adjusted P value < 0.01 The mRNA, lncRNA, and miRNA expression profiles were screened out. There were 193 nodes (199 mRNAs obtained were subjected to differential expression and 6 lncRNAs) and 229 edges in the co-expression analysis. The screening thresholds for mRNA and network of mRNA-lncRNA, where lncRNA Pvt1 miRNA were adjusted P value < 0.01 and |logFC| > 2, oncogene (PVT1) had the highest connectivity degree and for lncRNA was adjusted P value < 0.05 and (Figure 3A). In the co-expression network of mRNA- |logFC| > 1. A total of 1476 (323 upregulated and 1153 miRNA, there were 184 nodes (392 mRNAs, 102 downregulated) DEMs, 9 (5 upregulated and 4 miRNAs) and 1,935 edges (Figure 3B). www.aging-us.com 8711 AGING Figure 1. Heatmaps of differential expression mRNAs (top 10), lncRNAs, and miRNAs (top 10). Red represents high expression and green represents low expression. www.aging-us.com 8712 AGING miRNA prediction carbon metabolic process and reciprocal meiotic recombination. A total of 200 lncRNA-miRNA pairs (3 lncRNAs and 141 miRNAs) were predicted, based on the lncRNA The mRNAs in the miRNA-mRNA relation pairs involved in lncRNA-mRNA co-expression pairs using obtained above were used as the target genes of miRNAs. starbase (Figure 4A). Additionally, the mRNA involved Since there were few target genes and enrichment in lncRNA-mRNA co-expression pairs were subjected analysis could not be carried out, thus the functions of to mRNA-miRNA prediction, and 117,570 pairs of each target gene were inquired on the genecards to obtain miRNA-mRNA (199 mRNAs and 2498 miRNAs) were the functions of all miRNA (data not shown). obtained with Score = 1 and Position = CDS. These mRNA-miRNA pairs were intersected with the obtained ceRNA