Identification of potential gene and microRNA biomarkers for colon cancer by an integrated bioinformatical approach
Original Article

Identification of potential gene and microRNA biomarkers for colon cancer by an integrated bioinformatical approach

Qingxia Ma1*, Jingyi Song2*, Zhen Chen2, Lei Li2, Bin Wang2, Ningning He2

1Department of Pharmacology, School of Pharmacy, 2School of Basic Medical Sciences, Qingdao University, Qingdao 266021, China

Contributions: (I) Conception and design: N He, B Wang; (II) Administrative support: B Wang; (III) Provision of study materials or patients: Q Ma, J Song, Z Chen; (IV) Collection and assembly of data: Q Ma, J Song, L Li; (V) Data analysis and interpretation: N He, B Wang; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

*These authors contributed equally to this work.

Correspondence to: Bin Wang. School of Basic Medical Sciences, Qingdao University, 38 Dengzhou Road, Qingdao, 266021, China. Email:; Ningning He. School of Basic Medical Sciences, Qingdao University, 38 Dengzhou Road, Qingdao, 266021, China. Email:

Background: A unique resource for genomic studies is the NCI-60 cancer cell line panel, which provides various publicly available datasets, including gene and microRNA (miRNA) transcripts, whole-exome sequencing, DNA copy number, and protein levels against 59 cancer cell lines. In this study, we aimed to identify differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) for colon cancers by analyzing NCI-60 gene and miRNA expression profiles.

Methods: The real-time quantitative RT-PCR (qRT-PCR) was used to validate the identified candidate genes and miRNAs. The functional enrichment analysis and pathway analysis were performed for DEGs and the network analysis was performed for predicted miRNA targets of DEMs.

Results: Finally, we integrated DEGs and DEMs and constructed a network, revealing the relationship for 5 miRNAs and 104 gene probes. The identified specific genes, miRNAs and integrated network for colon might provide new clues on possible mechanisms for colon cancer.

Conclusions: In conclusion, integrated bioinformatics approaches on publicly available datasets play critical roles for finding candidate biomarkers and potential mechanisms for cancers.

Keywords: NCI-60; differentially expressed genes (DEGs); differentially expressed miRNAs (DEMs); colon; network analysis

Submitted Aug 25, 2017. Accepted for publication Dec 05, 2017.

doi: 10.21037/tcr.2017.12.09


The NCI-60 is a panel of 60 human tumor cell lines which isolated from diverse histologies and 9 different tissues of origin. It mainly contains cancers of colorectal (CO), ovarian (OV), melanomas (ME), lung (LC), breast (BR), and central nervous system (CNS) origin (1). The NCI-60 panel was used by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI) of the USA in 1990. It is reported that NCI-60 is a comprehensively panel of diverse cell types at the DNA, RNA, protein, mutation, functional, and pharmacological levels (2-5). Now NCI-60 has been widely used in cancer research and bioinformatics for analysis cell phenotypes and pathway relationships (6).

Microarray is a powerful tool for detecting the expression pattern of RNA levels, including those of mRNA and microRNAs (miRNA) (7,8). Due to the wide use of microarray, a large number of microarray data have been collected and used to discover mechanism for tumorigenesis, development and therapy. Bioinformatical approaches are crucial for discovering more valuable information included in these datasets, particularly signaling pathways, complex biological processes and the interaction network of differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) (9,10).

Colon cancer is the fourth-leading cause of cancer-related death in the world. Despite after surgical resection of colon cancer, more than 50% of patients die of developing recurrence and disease relapse after several months (11,12). Clinical diagnosis of colon cancer relatively lagged behind dues to a serious threat to human health. Therefore, it is necessary to discover more effective biomarkers and reveal prediction molecular mechanisms of clinical significance of biomarkers.

Recently, construction of biophysics network models of numerous components has contributed to our understanding of the relationship between different types of biological molecules (13). In this study, we used the simple Pearson correlation coefficient (PCC) of the gene and miRNA expression profile over diverse NCI-60 cell lines. It is possible to obtain a certain number of significant correlations between gene expression and miRNAs on subsets of cancer samples.

In this research, our goal is to identify the DEGs and DEMs for colon cancer, functional enrichment analysis for DEGs, construct miRNA network with their target genes and gene-miRNA correlation network, which provide new clues on possible mechanisms for colon cancer.


Acquisition of microarray data

Microarray gene expression and miRNA expression data were obtained from the NCBI Gene Expression Omnibus (GEO) ftp ( NCI-60 gene expression data (GSE32474) and miRNA expression data (GSE26375) were retrieved. Gene probes or miRNAs with a fold change of >4 and P<0.01 for colon cancer over the median value were selected for the further analysis.

Functional analysis and pathway enrichment analysis

DAVID ( is an online tool that can be utilized to perform a functional analysis and pathway enrichment analysis for discovering the relationships among the selected gene sets (14). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed by the DAVID online program.

Analysis of miRNA-target mRNA network

The selected DEMs were imported to TargetScan (Release 7.1, which can search the target genes based on the conserved sites matching the seed regions of miRNAs. Cumulative weighted context++ score ≤−0.4 were set as the threshold. The miRNA-target mRNA network was constructed by Cytoscape ( (15).

Constructing gene-miRNA network

PCC and its P value between genes and miRNAs were calculated using gene and miRNA expression data from NCI-60 cell lines. The criteria of PCC >0.7 or PCC <−0.7 and P value <0.01 were applied to select gene-micorRNA relationship for further analysis. Finally, the network was constructed for links between compounds and genes using Cytoscape ( (15). The force-layout algorithm was applied to optimize the topology of the network.

Cell culture

HCT-116, HCT-8, A549 and H460 cell lines were all purchased from Cell Bank of Shanghai Institute of Biochemistry. HCT-116 and HCT-8 cells were cultured in DMEM/High Glucose (HyClone) medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/L streptomycin. A549 and H460 cells were cultured in RPMI 1640 medium (HyClone) with 10% FBS, 100 U/mL penicillin, and 100 mg/L streptomycin. All the cells were cultured under 37 °C and 5% CO2.

Quantitative real-time PCR

Total RNA was extracted from cells and tissues using TRI Reagent RT (Invitrogen, Carlsbad, CA, USA). Total RNA solution was stored at −70 °C. One µg total RNA was converted to cDNA using Stem-loop RT primers for miRNA and using anchored oligo (dT) primers for the gene. Following the manufacturer’s instructions, 2 µL cDNA was performed to real-time qPCR using SYBR® Green PCR Kit. Beta-actin was used as a loading control for gene while U6 for miRNA. The sequences of primers used in qRT-PCR were as showed in Table 1. All reactions had three replicates. The data were analyzed using the comparative 2−ΔΔCT method.

Table 1
Table 1 Primer sequences of DEMs and DEGs used in qRT-PCR
Full table

Software support and statistical analysis

Hierarchical clustering of gene expression profile was carried out using QCanvas (16). All images were formatted for optimal presentation using Adobe Illustrator CS4 (Adobe Systems, San Jose, CA, USA). To determine statistical significance, P value from t-statistic was calculated.


Identification of DEGs

To identify novel gene biomarkers for colon cancer, the DNA microarray of the NCI-60 cell line panel (GSE32474) was analyzed. A total of 560 gene probes were identified with significant differential expression in colon cancers. Among them, 285 gene probes with higher expression level and 265 gene probes with lower expression levels in colon cancer cells [log2 (fold change) >2 or log2 (fold change) <−2, P<0.01]. The up-regulated and down-regulated genes (DEGs) were used to generate the heatmap profile (Figure 1A). The differential expression pattern for two down-regulated genes (SPG20 and NR3C1) and two up-regulated genes (S100A14 and KLK6) were showed in Figure 1B.

Figure 1 Expression profile of the DEGs for colon cancer cell lines across NCI-60 cancer cell lines. (A) A heatmap showed 285 upregulated and 265 downregulated genes. Red, upregulation; green, downregulation; (B) SPG20, NR3C1, S100A14 and KLK6 showed a colon-specific expression pattern. DEGs, differentially expressed genes.

GO function and KEGG pathway analyses

To explore the functions of those DEGs in depth, the biological process, pathway annotation and molecular function were revealed using DAVID Gene Functional Classification Tool. The GO analysis revealed that the DEGs were significantly involved in the gap junction assembly, neural tube closure, positive regulation of cell migration, wound healing and extracellular matrix organization (the top 5 biological process) (Figure 2A). Furthermore, the KEGG pathway was used to determine the pathways involved in colon cancers. The result showed that DEGs were primarily involved in pathways in cancer, PI3K-AKT signaling pathways, cell adhesion, focal adhesion, tight junction and etc. (Figure 2B). The results collectively indicated that genes involved in the cell proliferation and migration were significantly associated with colon cancers.

Figure 2 GO and KEGG analyses of DEGs for colon cancer cell lines. (A) The top five terms of molecular functions, cell components and biological processes of GO enrichment analysis in DEGs; (B) KEGG pathway analysis of the DEGs. *, P<0.05; **, P<0.01. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes.

Experimental validation of the key DEMs

According to the transcriptomic data and bioinformatics analysis, seven genes were selected as potential gene biomarker candidates, including KLK6, S100A14, ANO1, LYZ, ESRP1, ACSLC5 and FOXQ1, and their roles in colon cancer have not been clearly addressed before. The previous study has been showed that KLK6 may represent a potential unfavorable prognostic biomarker for colon cancer (17). Overexpression of S100A14 in colon tumors may have a potentially important function in malignant transformation (18). These seven genes were further verified by qRT-PCR. As showed in Figure 3, the expression level of ANO1, LYZ, ESRP1, ACSLC5, FOXQ1, KLK6 and S100A14 were significant highly expressed in colon cancer cells and colon tissues.

Figure 3 Experimental validation of the key DEGs. The qPCR analysis of ANO1 (A), LYZ (B), ESRP1 (C), ACSL5 (D), FOXQ1 (E), KLK6 (F) and S100A14 (G) expression level in colon cancer cells (HCT-8 and HCT-116), colon cancer tissue samples (CO-tissue 1 and CO-tissue 2), lung cancer cells (H460 and A549) and lung cancer tissue samples (LC-tissue 1 and LC-tissue 2). The data represent mean ± SEM (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. DEGs, differentially expressed genes; qPCR, quantitative polymerase chain reaction; CO, colorectal; LC, lung cancer; SEM, standard error of measurement.

Identification of DEMs and miRNA-target mRNA network

There were 27 miRNA probes were identified from GSE26375 for colon cancers [log2 (fold change) >2 or log2 (fold change) <−2, P<0.01] ( Figure 4A). Especially, five up-regulated miRNAs (miR-7, miR-200a, miR-200b, miR-200c, miR-141) were identified. Those five miRNAs were further verified by qRT-PCR (Figure 4B,C,D,E,F). Interestingly, miR-200a, miR-200b, miR-200c, and miR-141 were members of miR-200 protein family (19). Those five DEMs were imported to TargetScan (Release 7.1, which can search the target genes based on the conserved sites matching the seed regions of miRNAs. Cumulative weighted context++ score ≤−0.4 were set as the threshold. The miRNA-target mRNA network was constructed by Cytoscape (Figure 5).

Figure 4 The microRNA expression profile for colon cancers. (A) DEMs for colon cancers. (B-F) Experimental validation of the key DEMs. The qPCR analysis of miR-7 (B), miR-200b (C), miR-200a (D), miR-429 (E) and miR-200c (F) expression level in colon cancer cells, colon cancer tissue samples (CO-tissue), lung cancer cells and lung cancer tissue samples (LC-tissue). The data represent mean ± SEM (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. DEMs, differentially expressed genes; qPCR, quantitative polymerase chain reaction; CO, colorectal; LC, lung cancer; SEM, standard error of measurement.
Figure 5 The network of microRNA and its predicted target genes. A network consisted of miR-7, miR-200b/c, miR-200a/141 and their target genes. The selected DEMs were imported to TargetScan (Release 7.1, which can search the target genes based on the conserved sites matching the seed regions of miRNAs. Cumulative weighted context++ score −0.4 were set as the threshold. The miRNA-target mRNA network was constructed by Cytoscape. DEMs, differentially expressed genes.

Construction of gene-miRNA network

To further explore the interactions of DEGs and DEMs, the PCC and its P value between genes and miRNAs were calculated using gene expression and miRNA expression data on 59 NCI-60 cell lines. The criteria of absolute PCC >0.7 and P value <0.01 were applied to select gene-miRNA relationship for further analysis. Finally, the network for a total of 104 gene probes and 5 miRNAs was constructed using Cytoscape (Figure 6). The data provide a useful resource for studying and predicting the relationship of genes and miRNAs for colon cancer.

Figure 6 Correlation network of gene probes and microRNA. The correlation between gene expression and microRNA expression was calculated across the NCI-60 cell lines. Totals of 104 gene probes and 5 microRNAs were selected with a significant correlation displayed with connecting edges.


Despite advance in medical and surgical therapy, the colon cancer is the third common diseases in the world, moreover, in developed countries, colon cancer has a higher incidence than developing countries (20). In developed countries, more than 65% of diseases are colon cancers (21). The characteristics of colon cancer are invasive and spread to other tissues of the human body. The lethality of lethal malignancy globally is due to the difficulties in detecting colon cancer at an early state. Therefore, it is essential and beneficial to figure out the etiological factors and mechanisms of colon cancer to improve prevention and survival rate (22). Recently, microarray technology has been an effective tool for revealing the expression of general genetic alteration in different physiological and pathological status, which enables the discovery of new targets for diagnosis, therapeutic, and prognosis of malignant cancers.

In this study, a total of 560 gene probes, including 285 significantly up-regulated genes and 265 significantly down-regulated genes were identified used NCI-60 microarray dataset. These differential expressed genes mainly were enriched in cell proliferation, migration, invasion, apoptosis and survival rate. Among those genes, seven genes were selected as potential gene biomarker candidates, including KLK6, S100A14, ANO1, LYZ, ESRP1, ACSLC5 and FOXQ1. Anoctamin-1 (ANO1) as transmembrane protein 16A (TMEM16A) was found in a number of cancers, such as gastrointestinal stromal tumor (GIST), BR cancer (23,24). Moreover, ANO1 plays an important role in cell proliferation, tumorigenesis and progression (25). Recent studies shown that in various cell types of ANO1 could activate calcium-activated chloride channels (CaCCs), molecular and electrophysiological studies indicated that ANO1 plays a critical role in metastatic tumor (26,27). Kallikrein-related peptidase (KLK) is a member of the human kallikrein gene family, in length, KLK6 encodes of 224 amino acid with trypsin-like activity (28). In addition, the KLK6 gene was validated to be a secreted protein which shares significant homologies with other kallikreins and the enzyme (29). As a new potential biomarker, the expression of KLK6 was significantly increased in patients with cancer in an late-stage, accumulating evidence demonstrates that KLK6 may play a role in the development and progression of cancer (30). S100A14 is one protein of S1004 proteins. It has been reported that play a role in cancer cells invasion and metastasis (31). S100A14 protein exert its function which regulates tumor invasion by modulating the level of matrix metalloproteinase MMP-1 and MMP-9 (32). In addition, S100A14 could regulate the expression of MMP-2 and p-53 dependent pathway to affect tumor cells invasion (33). As a member of the long-chain acyl-CoA synthetase (ACSL) gene family, ACSL5 is overexpression on tumor versus other members of ACSL family (34,35). As a unique position among the ACSL gene family, ACSL5 at chromosome 10q25.1–q25.2, mainly located on mitochondria and regulated apoptosis of cells (36). While, human ACSL5 exert activation usually through certain apoptosis pathway (37).As a factor associated with metastatic, epithelial splicing regulatory protein 1 (ESRP1), is an RNA-binding proteins (RBPs) and splicing factor (38). ESRP1 have ability to bind the 5’UTR of a wide range of cancer-related genes and alter their translation to exhibit it functions, as a tumor expression ESRP1 inhibit epithelial to mesenchymal transition (EMT) in various cancers (39-42). However, in the present study, in cancer cells over-expressing of ESRP1 cloud enhance their metastatic potential (43). Moreover, metastatic progression of carcinoma cells is a positive association with the level of CD44v6 isoform, a target of ESRP1 (44). And ESRP1 could target growth factor receptor including FGFR1/2 pathway, AKT signaling and Snail activation to promote cancer progression (38).

miRNA, a type of small non-coding RNA molecule which consists of 18–25 nucleotides. It can regulate target gene expression to affect cell biological process such as cell apoptosis, cell proliferation, cell differentiation, and cell metastatic (45). Increasing evidence demonstrated that some miRNA can affect the pathogenesis of various cancers, including colon cancer. Therefore, miRNA may be biomarker for tumor diagnosis and treatment (46). In the present study, we identified 27 DEMs for colon cancer cells. Especially, five up-regulated miRNAs (miR-7, miR-200a, miR-200b, miR-200c, and miR-141) were identified. MiRNA-7 plays a pivotal roles on tumors growth and metastatic by affecting the region of focal adhesion kinase (FAK) mRNA, which inhibit the level of FAK protein (47). In cancer cells, miRNA-7 inhibits the expression of FAK, FAK is mainly located at the cell cytoplasm and promotes the secretion of MMPs, and therefore, miRNA-7 may be targeting the level of MMPs to exert its function (48). MiRNA-200 family consisting of five members (miRNA-200a, miRNA-200b, miRNA-200c, miRNA-141 and miRNA-429) which participating in the progression of EMT (49). As a member of miRNA-200, miRNA-429 expression in colon cancer was significantly up-regulated compared with LC, as reported, miRNA-429 have the ability to inhibit loss of E-cadherin undergoing EMT (50). Similar results that, miRNA-200a and miRNA-200b were up-regulated in colon cancer cells and tissue, moreover, miRNA-200a and miRNA-200b showed higher expression in cancer tissue than normal tissue which were identified as candidate biomarkers in tumor (51,52).

In summary, data integration and data mining play critical roles for finding the candidate biomarkers and the mechanisms of cancers. In this study, we analyzed a total of 560 DEGs and 27 DEMs. Some significant miRNAs and genes were validated by qRT-PCR, including ANO1, KLK6, A100A14, ACSL5, ESRP1 genes and miRNA-7, miRNA-200b, miRNA-200a, miRNA-200c and miR-141. Nevertheless, these identified genes and miRNA need to be confirmed by more researchers in the future, our study could provide new methods for diagnosis and treatment of colon cancer patients.


Funding: This study was funded by the National Nature Science Foundation of China [81602621] and Postdoctoral Applied Research Programs of Qingdao [2016061].


Conflicts of Interest: The authors have no conflicts of interest to declare.

Ethical Statement: The clinical samples were obtained with informed consent, and the study protocol was approved by the Ethics Committee of the Affiliated Hospital of Medical College Qingdao University.


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Cite this article as: Ma Q, Song J, Chen Z, Li L, Wang B, He N. Identification of potential gene and microRNA biomarkers for colon cancer by an integrated bioinformatical approach. Transl Cancer Res 2018;7(1):17-29. doi: 10.21037/tcr.2017.12.09