Transcriptome-scale analysis of functional alternative back-splicing events in colorectal cancer

Circular RNAs (circRNAs) are a class of non-polyadenylated RNAs generated from back-splicing of genes. Multiple circRNAs can be generated at a single gene locus through alternative back-splicing events (ABS), sharing the same 5' or 3' back-splice site. To date, how prevalent ABS events are and how they are participated in carcinogenesis of human colorectal cancer (CRC) remains unexplored.

To explore the functional roles of ABS events in CRC carcinogenesis, we analyzed ribosomal RNA-depleted transcriptome sequencing data of 176 CRC samples and characterized the landscape of ABS events in CRC. CRC cancer-related ABS events were identified by comparing paired CRC tumor tissues and adjacent normal tissues. Then, univariate and multivariate Cox regression was used to find prognostic ABS events. Moreover, in vitro and in vivo assays were used to exploring the functional roles of circXPO1-1 and circXPO1-2 in CRC.

We totally identified 19,611 high confidence circRNAs in CRC, among which 17,874 (91·1%) of circRNAs were found recurrently. The number of ABS circRNAs accounted for 68.8% of all identified high confidence circRNAs, which suggested that ABS events are prevalent in CRC transcriptome. Particularly, 552 ABS circRNAs were found to be aberrantly expressed between paired CRC tumor tissues and adjacent normal tissues, and their parent genes are closely associated with cancer-related hallmarks. In addition, 13 differential ABS circRNAs were identified to be associated with CRC patient survival and could act as independent prognostic indicators. Furthermore, we identified two ABS circRNAs of XPO1 gene (circXPO1-1 and circXPO1-2). The result showed that overexpression of circXPO1-2 inhibited CRC cell proliferation, migration, and invasion in vitro and in vivo, whereas circXPO1-1 is not, indicating that the circularization isoforms of XPO1 gene have different functions in CRC.

In conclusion, our work provides the landscape of ABS events in CRC transcriptome and the close association of ABS circRNAs with tumorigenesis offers a new set of targets with potential clinical benefit.