High expression of circRNAs in muscle and stromal cell confound bulk tissue analyses in cancer research [RNA-Seq]
Circular RNAs (circRNAs) are covalently closed molecules that are likely to play important roles in cancer development and progression. Hundreds of differentially expressed circRNAs between tumors and adjacent normal tissues have been identified in studies using RNA-sequencing or microarrays, emphasizing a strong translational potential. However, previous studies were performed using RNA from bulk tissues and the spatial expression patterns of most circRNAs are unknown. Here, we show that the majority of differentially expressed circRNAs from bulk tissue analyses of colon tumors relative to adjacent normal tissues are, surprisingly, not differentially expressed when comparing cancer cells directly with normal epithelial cells. By manipulating the proliferation rates of cells grown in culture, we find that these discrepancies are explained by circRNAs accumulating to high levels in quiescent muscle cells due to their high stability and, on the contrary, circRNAs are diluted to low levels in the fast-proliferating cancer cells due to their slow biogenesis rates. Thus, we observed striking changes in circRNA expression patterns within different sub-compartments of colon tumors and adjacent normal tissues. Likewise, we find that the high circRNA content in muscle cells can be a strong confounding factor in bulk analyses of circRNAs in bladder and prostate cancer. Together our findings emphasize the limitations of using bulk tissues for studying differential circRNA expression in cancer and highlight a particular need for spatial analysis in this field of research. Overall design: In this study, 2 cell lines (Caco2 and hASMC) were interrogated for their circRNA levels before and after starvation. Four different timepoints: 24h: 24h after seeding with complete media; 96h: 24h of seeding with complete media follwed by 72h in starvation (no FBS in media); 120h: 24h of seeding with complete media followed by 96h in starvation (no FBS in media); 168h: 24h of seeding with complete media followed by 96h in starvations and followed by 48h with complete media (Recovered).