In Depth Functional Analysis of Circular RNAS in Regulating Chemoresistance in Colorectal Cancer

Abstract

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. An increase in yearly CRC cases has made CRC the second highest contributor to cancerrelated mortality rates both globally and in Malaysia. Surgery, chemotherapy and radiotherapy remain the main treatment management options for patients. FOLFOX, a 5-fluorouracil (5-FU) based chemotherapy is the standard of care for adjuvant colorectal cancer (CRC) patients. However, it is widely acknowledged that disease recurrence due to chemoresistance remains a major clinical limitation. Thus, there is an urgent need to identify a novel biotarget to predict chemoresistance in CRC and understand the chemoresistance mechanism. Circular RNAs (circRNAs) are a novel group of endogenous non-coding RNA molecules that have a distinctive characteristic of noncanonical splicing without a 5’ cap and 3’ poly-A tail. They are generated by nonsequential back splicing or pre-mRNA transcripts and are found to be more stable thus insusceptible to exonucleases when compared to their linear counterparts. Besides, they act as microRNA (miRNA) sponges that regulate gene expression specifically at every stage of cell and disease development. Nevertheless, their role in the development of CRC particularly in chemoresistance is still largely unknown. Therefore, this research aims to identify differentially expressed circRNAs in chemotherapy and to investigate the role of circRNA and miRNA in regulating chemoresistance. This study used chemotherapy responder and non-responder fresh frozen tissues to perform high throughput microarray. Using fold change ≥ 1.5 and p < 0.05, our findings revealed that 131 circRNAs were upregulated and 144 downregulated in the non-responder tissue group compared with the responder tissue group. Based on discovery and validation data, we found that circANAPC7 (hsa-circRNA-101144) was significantly upregulated in the non-responder tissue group and resistant CRC cell lines. Functionally, the study showed that a decrease in circANAPC7 significantly decreased CRC proliferation, migration, invasion, drug resistance and caused cell cycle arrest. Mechanistically, by performing RNA pull-down assay and dual reporter luciferase assay, circANAPC7 was identified to sponge hsa-miR-338-3p. Overexpression of hsa-miR-338-3p was able to reverse the negative effect of circANAPC7 induced malignant phenotypes. This research also found that KRT23 was a direct target to hsa-miR-338-3p and had a higher expression level in the non-responder group. Taken together, the findings have produced specific circRNA profiles in the chemotherapy resistant group. The data indicated that circANAPC7 can potentially be a prognostic marker in understanding CRC chemotherapy resistance. The study has demonstrated that the therapeutic target of circANAPC7/hsa-miR-338-3p interaction indirectly facilitates the expression of KRT23 which may be a promising treatment target for CRC patients.