Abstract
BackgroundDespite the clinical significance of liver metastases, the difference between molecular and cellular changes in primary colorectal cancers (CRC) and matched liver metastases is poorly understood.MethodsIn order to compare gene expression patterns and identify fusion genes in these two types of tumors, we performed high-throughput transcriptome sequencing of five sets of quadruple-matched tissues (primary CRC, liver metastases, normal colon, and liver).ResultsThe gene expression patterns in normal colon and liver were successfully distinguished from those in CRCs; however, RNA sequencing revealed that the gene expression between primary CRCs and their matched liver metastases is highly similar. We identified 1895 genes that were differentially expressed in the primary carcinoma and liver metastases, than that in the normal colon tissues. A major proportion of the transcripts, identified by gene expression profiling as significantly enriched in the primary carcinoma and metastases, belonged to gene ontology categories involved in the cell cycle, mitosis, and cell division. Furthermore, we identified gene fusion events in primary carcinoma and metastases, and the fusion transcripts were experimentally confirmed. Among these, a chimeric transcript resulting from the fusion of RNF43 and SUPT4H1 was found to occur frequently in primary colorectal carcinoma. In addition, knockdown of the expression of this RNF43-SUPT4H1 chimeric transcript was found to have a growth-inhibitory effect in colorectal cancer cells.ConclusionsThe present study reports a high concordance of gene expression in the primary carcinoma and liver metastases, and reveals potential new targets, such as fusion genes, against primary and metastatic colorectal carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2596-3) contains supplementary material, which is available to authorized users.
Highlights
Despite the clinical significance of liver metastases, the difference between molecular and cellular changes in primary colorectal cancers (CRC) and matched liver metastases is poorly understood
CDNA library preparation and high-throughput pairedend RNA sequencing Total RNA was isolated from fresh-frozen tissues of the conditioned volunteers and patients (NC, normal colon; PC, primary colon carcinoma; LM, colon-liver metastases; NL, normal liver) using TRIzol reagents (Invitrogen, USA), and subsequently treated with RNase-free DNaseI for 30 min at 37 °C, to remove residual DNA
The hierarchical clustering results showed that normal colon and liver were successfully distinguished from colorectal carcinoma, but primary carcinoma preferentially clustered with their matched liver metastases (Fig. 1). These results suggest a high concordance of gene expression in the primary carcinoma and liver metastases
Summary
Transcriptome sequencing and mapping Five sets of quadruple-matched tissues (primary carcinomas, liver metastases, normal colon, and liver) were collected from Pusan National University Hospital. Functional enrichment analysis of differentially expressed genes The common 1895 DEGs in primary carcinoma and liver metastases, compared with normal colon, were detected. 3.4E-2 tumors (FDR < 0.05, fold change > 2) Of these genes, we selected 14 DEGs compared with normal colon (FDR < 0.05, fold > 2) and normal liver tissues (FDR < 0.05, fold >2) (Additional file 6: Table S3). ZMYND8-SEPT9 and ACE2-PIR fusion transcripts were successfully amplified by RT-PCR, and these fusion junctions were confirmed by Sanger sequencing (Additional file 7: Figure S3) These results confirmed fusion events in the sample, consistent with results of RNA-seq analysis. In the HT29 cell line, cell proliferation decreased at 72 h after transfection (Fig. 4f ) These results suggest that the RNF43-SUPT4H1 fusion transcript has a positive effect on cell growth in colorectal cancer
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