KRAS-dependent Regulation of Extracellular RNAs in Colorectal Cancer

Abstract

There is growing evidence for the regulatory roles of extracellular RNAs (exRNAs) in mediating cell-to-cell communication. To test whether exosomal RNA might also contribute to changes in gene expression in recipient cells, and to test whether mutant KRAS might regulate the composition of secreted RNAs, we comprehensively profiled small and long RNAs of cells and matched exosomes from isogenic colorectal cancer (CRC) cell lines differing only in KRAS status by RNA sequencing. We found that exosomal profiles are distinct from cellular profiles, and differentially enriched for specific small RNA, circular RNA (circRNA) and long RNA transcripts in exosomes dependent on KRAS status. Our small RNA analysis found that miR-10b was selectively increased in wild type KRAS exosomes while miR-100 was increased in mutant KRAS exosomes. In Transwell co-culture experiments, mutant KRAS donor cells conferred miRNA-mediated target repression in wild type KRAS recipient cells. In addition, we developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. We found a significant down-regulation of circRNAs at a global level in mutant KRAS cells compared to wild type KRAS cells, indicating a widespread effect of mutant KRAS on circRNA abundance. Interestingly, circRNAs were more abundant in exosomes than cells, independent of KRAS status. Our long RNA analysis revealed that distinct RNAs species, such as pseudogene and antisense transcripts, are enriched in exosomes compared to cellular profiles. Additionally, specific mRNAs, such as Rab13, are upregulated in mutant KRAS exosomes. Here, we present comprehensive data to identify the broad and diverse classes of extracellular RNAs secreted in exosomes and we demonstrate that export of specific RNA can be altered by oncogenic KRAS signaling to potentially function beyond the cell of origin. Collectively, this will advance our understanding of exRNA biology in CRC and facilitate the development of potential exRNA biomarkers.