Introduction
Intrahepatic cholangiocarcinoma (ICC) is the second most frequently occurring primary liver cancer with a high mortality rate (1), which affects 1–2 per 100,000 patients (2). Various factors have previously been suggested to contribute to the progression of ICC, including sclerosing cholangitis and hepatobiliary flukes (3). Ultrasound scans may provide diagnostic information (4); however, specific diagnostic criteria for patients with ICC remain to be elucidated. Therefore, numerous patients present with ICC for diagnosis at an advanced stage, which may contribute to its poor prognosis (5). It is therefore important to identify potential biomarkers of ICC to aid prevention and identification of therapeutic strategies.
The progression of ICC is associated with numerous genetic factors, including gene mutations and dysregulation of gene expression (6). Various technological advances have identified molecular targets. Weber et al (7) demonstrated that low frequency alterations of phosphatase and tensin homolog, cyclin-dependent kinase inhibitor 2A, and breast cancer 1/2 may explain the mutation spectrum in ICC, based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-based targeted somatic multiplex-mutagenesis. Lee et al (8) profiled the expression of 315 genes and screened 400 alterations in 84 genes, via hybridization capture, which may act as therapeutic targets of ICC (8). Various drugs or therapeutic methods have been developed based on the aforementioned identified biomarkers. Lovastatin, which is a 3-hydroxy-3-methylglutaryl-coenzyme-CoA reductase inhibitor, may inhibit the proliferation, migration and adhesive activities of ICC cells, by inhibiting the expression of transforming growth factor-β1, cyclooxygenase-2, and intracellular adhesion molecule-2 (9). Furthermore, various genes have previously been demonstrated to affect drug sensitivity and resistance in ICC. Tepsiri et al (10) demonstrated that the expression of multidrug resistance-associated protein 3 was significantly associated with the half maximal inhibitory concentration of etoposide in ICC. Various biomarkers have been identified; however, the underlying mechanism remains to be fully elucidated, to improve the prognosis and therapy of ICC.
Gene microarrays profile the expression of thousands of genes simultaneously and have been incorporated in numerous cancer and ICC associated investigations (11–13). The present study compared gene and microRNA (miRNA/miR) expression data in ICC samples with healthy liver tissues, and differentially expressed genes (DEGs) and miRNAs (DEMs) were identified. Functional and pathway analyses were conducted, and subsequently, a miRNA-gene regulation network was constructed to explore potential biomarkers of ICC to improve its prognosis.
Discussion
In recent years, the emergence of novel diagnostics and treatments, including B-mode ultrasound and chemotherapy, has improved the prognosis of ICC. However, limitations are present in terms of its early diagnosis and cure. The present study conducted a statistical comparison of mRNA and miRNA expression profiles between ICC and healthy liver tissues samples, in order to identify DEGs, DEMs and ASGs. Functional and pathway analyses of DEGs were conducted to explore associated functions and pathways. The miRNA-gene regulation network was constructed to identify the potential biomarkers of ICC.
The GeneChip Human Gene 1.0 ST array (HuGene-1_0-st-v1) is a transcript-based array, used for the detection of gene expression values, and estimation of AS based on the well-annotated exon probes (22). The present study identified the DEGs and ASGs in ICC samples compared with healthy liver tissues, based on the data detected by HuGene-1_0-st-v1. Functional enrichment analysis indicated that the DEGs were primarily involved in the biological processes associated with cell activity, including cell adhesion and regulation of cell proliferation, which have previously been demonstrated to be associated with cancer progression (23). Pathway enrichment analysis revealed that DEGs were significantly enriched in the pathways associated with substance metabolism and degradation, including fatty acid, glycine, serine and threonine metabolism, and valine, leucine and isoleucine degradation. The extracellular matrix-receptor pathway was also demonstrated to be enriched in DEGs, consistent with the findings of Lee et al (24). The genes that were simultaneously presented in the extracellular matrix-receptor pathway and were revealed to be ASGs, included chondroadherin (CHAD), integrin subunit (ITG)-A3, ITG-B1 and laminin subunit alpha-5 (LAMA5). Three of the aforementioned genes, (ITGA3, ITGB1 and LAMA5) have previously been demonstrated to be associated with the progression of ICC (25,26). CHAD encodes a cartilage matrix protein, which may mediate adhesion of isolated chondrocytes. CHAD may be included in the phosphoinositide 3-kinase-Akt and focal adhesion signaling pathways, and may be regulated by p53, which is associated with the pathogenesis and development of numerous cancers, including ICC (http://www.genecards.org). Therefore, CHAD may be a novel biomarker of ICC that may contribute to its progression; however, further studies are required to confirm this.
miRNA are non-coding RNA molecules, which contain ~22 nucleotides, and are present in animals, plants and viral genomes. miRNAs may induce RNA-silencing and regulate gene expression at the post-transcriptional stage (27,28). The dysregulation of miRNA may result in numerous diseases, including ICC (29–31). The present study identified DEMs in ICC samples compared with healthy liver tissues samples, and the screening of DEM target genes was conducted using the TargetScan database. A miRNA-gene regulation network was constructed based on the DEMs and the overlapping genes among the DEGs, ASGs and target genes of the DEMs. In the regulation network, hsa-miR-96 regulated 22 target genes and 6 out of these regulation pairs [hsa-miR-96-tumor protein P63 regulated 1, hsa-miR-96-sodium voltage-gated channel alpha subunit 9, hsa-miR-96-lon peptidase 2, peroxisomal, hsa-miR-96-T-cell lymphoma invasion and metastasis 1 (TIAM1), hsa-miR-96-chloride voltage-gated channel 5, hsa-miR-96-glycerol kinase] revealed an opposite trend in the alterations of miRNA and mRNA expression values (upregulated miRNA and downregulated gene expression levels). hsa-miR-96 is closely associated with cell proliferation and growth, and Collins et al (32) demonstrated that hsa-miR-96 may be used to identify cholangiocarcinoma and pancreatic adenocarcinoma. Furthermore, the dysregulation of hsa-miR-96 was revealed to contribute to the progression of ICC (33). TIAM1 is a target gene of hsa-miR-96, which is important in cellular migration and remodeling of the actin cytoskeleton, and may be involved in the proliferation and migration of ICC via effects on RasGEF domain family member 1A expression (34). Further target genes of hsa-miR-96 have previously been identified, and may act as novel biomarkers of ICC, however further experimental verification is necessary (35).
In conclusion, the present study identified DEGs, ASGs and DEMs in ICC samples compared with healthy liver tissues, via bioinformatics analysis of miRNA and mRNA expression data. Functional and pathway analyses indicated that DEGs were primarily involved in biological processes or pathways associated with cancer progression or substance metabolism. A miRNA-gene regulation network was constructed based on DEMs and overlaps among DEGs, ASGs and target genes of DEMs. Furthermore, biomarkers that have previously been identified, including hsa-miR-96 and TIAM1, and numerous novel biomarkers in ICC, including anthranilate synthase component II and sodium voltage-gated channel a subunit 9, were identified. However, the functions of these biomarkers in ICC remain to be elucidated.