Transcriptome analysis of paired primary colorectal carcinoma and liver metastases reveals fusion transcripts and similar gene expression profiles in primary carcinoma and liver metastases

Abstract

Background: 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.

Methods: In 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).

Results: The 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.

Conclusions: The 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.

Keywords: Colorectal cancer; Expression profiling; Gene fusion; RNA-seq.

Fig. 1

Hierarchical clustering of expression profiles. Data are presented in a matrix format, in which each row represents an individual gene and each column represents a different tissue sample. Red, high expression; green, low expression. NC, normal colon; PC, primary carcinoma; LM, liver metastases; NL, normal liver

Fig. 2

The Venn diagram for comparison of gene fusion events that are unique or shared in the 4 tissue types. NC, normal colon; PC, primary carcinoma; LM, liver metastases; NL, normal liver

Fig. 3

RNF43-SUPT4H1 fusion in validation sets. a schematic of RNF43, SUPT4H1 and the resulting RNF43-SUPT4H1 fusion transcript. b PCR and Sanger sequencing validation of the positive fusion samples in validation sets. NC, normal colon; PC, primary carcinoma; LM, liver metastases. c RNF43-SUPT4H1 fusion screening in colorectal cancer cell lines

Fig. 4

Knockdown of RNF43-SUPT4H1 fusion transcript results in decreased cell proliferation. Quantitative RT-PCR of original RNF43 (a) and SUPT4H1 (b) gene in the DLD-1 cell line after transfection of siRNA targeting the RNF43-SUPT4H1 fusion transcript. c and e, RT-PCR of RNF43-SUPT4H1 fusion transcript in the DLD-1 and HT29 cell line after siRNA treatment. d and f, Knockdown of RNF43-SUPT4H1 fusion transcript decreased cell proliferation in the DLD-1 and HT29 cell lines