Profiling of exosomal circular RNAs in folfox-resistant colon cancer cell line HCT116

Abstract

Chemo-resistance is a major challenge for FOLFOX chemotherapy in colorectal cancer (CRC). There is no clinical biomarker for early prediction of chemo-resistance in CRC, so development of novel biomarker is essential. Exosomes are nanovesicles secreted by living cells as circulating entities into body fluids. Exosomes are intercellular messengers which transport nucleic acids, proteins and lipids between cells. Circular RNAs (circRNAs) are discovered in exosomes. CircRNAs are stable with circular loop structure which is resistant towards exonuclease degradation. CircRNAs are involved in post-transcriptional regulation of gene expression by inhibiting miRNAs. Crosstalk between circRNAs in exosomes and chemo-resistance in CRC remains unknown. This research aims to identify exosomal circRNAs associated with FOLFOX-resistance in CRC. We developed a FOLFOX-resistant HCT116-R cell line (HCT116-R) based on parental HCT116 cells (HCT116-P). Exosomes from HCT116-R cells, HCT116-P cells and patient serum were characterized using transmission electron microscopy (TEM), dynamic light scattering and western blot. TEM confirmed translucent, cup-shaped morphology of our exosomes. Size distributions of our exosomes were between 65.74 nm and 328nm. TSG101 was expressed in all our exosomes while CD9 and CD63 were expressed in HCT116-R exosomes and serum exosomes. We performed circRNAs microarray using exosomal RNAs and cellular RNAs from HCT116-R and HCT116- P cells. We validated our microarray data using serum samples. We performed drug sensitivity assay and cell cycle analysis to characterize selected circRNA after knockdown. Using fold change >2 and p <0.05, 105 exosomal circRNA with significantly higher expression and 34 circRNAs with lower expression were reported in HCT116-R exosomes as compared to HCT116-P exosomes. We reported that exosomes transfer drug resistance and circRNAs from resistant cells to sensitive cells. Knockdown of circ_0000338 improved the chemo-resistance of CRC cells. We have proposed that circ_0000338 may have dual regulatory roles in chemo-resistant CRC. Exosomal circ_0000338 could be a potential biomarker for further validation in CRC.