RNA-Seq profiling of circular RNAs in human colorectal Cancer liver metastasis and the potential biomarkers

To identify circRNAs that were differentially expressed in CRC patients with liver metastasis, the secondary sequencing was used to profile circRNAs expression in the tissue samples from three CRC patients with liver metastasis and three matched CRC patients. The basic characteristics of the patients was provided in Additional file 1: Table S2. Under the sequencing, total 66,855 circRNAs were detected in the tissue samples. The list of total circRNAs expression profiling was shown in Additional file 1: Table S3. The scatter plot showed the variation of circRNAs expression level between CRC patients with or without liver metastasis (Fig. 1a). As was showen in volcano plot, the significant differentially expressed circRNAs between the two groups were identified with fold-changes of greater than 2.0 and p value less than 0.05 (Fig. 1b). From the total 66,855 circRNAs, 92 circRNAs were significant up-regulated and 21 circRNAs were significant down-regulated (Fig. 1c and Additional file 1: Table S4). According to the source of the circRNA formation, it revealed that most significant differentially expressed circRNAs were transcribed from the exons of coding protein (Fig. 1d). Further, we classified of all differentially expressed circRNAs base on the different location on the chromosomes. All chromosomes have the expression of circRNA, and we could found the differentially expressed circRNAs except Y chromosome (Fig. 1e). Hierarchical clustering demonstrates these significant differentially expressed circRNAs (Fig. 1f). Thereinto, the top 10 circRNAs with significant up-regulation or down-regulation were listed in Additional file 1: Table S5, of these 20 circRNAs, the fold-changes were at least greater than 10. The circRNA-seq data and analysis suggest that some cricRNAs expressed level were different in the CRC patients with liver metastasis compared with CRC patients.

Considering the expression level of down-regulated circRNAs in CRC-m patients were very low and not easy to detect in disease progression, we only focus on the up-regulated circRNAs. From the list in Additional file 1: Table S5, we first selected the 4 up-regulated circRNAs which had the most significantly different expression. To verify the circRNA-seq results of these 4 circRNAs, qRT-PCR was performed in the tissue samples from 16 CRC-m patients and 24 CRC patients. As shown in Fig. 2a, the circRNA_0001178 and circRNA_0000826 were significantly up-regulated in CRC-m patients, which were consistent with previous sequencing results.

From the validation, we confirmed the expression levels of circRNA_0001178 and circRNA_0000826 were increased in CRC-m patients. Then the ROC curve analysis was performed to assess the diagnostic value of these two circRNAs in CRC-m patients. The AUC was 0.945 (95% CI: 0.863–1.000 P < 0.001) for circRNA_0001178 (Fig. 2b) and 0.816 (95% CI: 0.656–0.977, P < 0.01) for circRNA_0000826 (Fig. 2c). The results suggested the potential diagnostic value for these two circRNAs in CRC-m patients.

We built a circRNA-miRNA-target gene network for circRNA_0001178 and circRNA_0000826 using Cytoscape respectively. In the network map, we showed the top 5 miRNA that potentially bind to the circRNA and the five most likely target genes to each miRNA (Fig. 2d and e). From this figure, we can clearly see the potential target of circRNA_0001178 and circRNA_0000826. It gave us a clear direction to study the specific mechanism of these two circRNA in the liver metastasis of colorectal cancer.