Tumor-suppressive circRNAs in colorectal cancer spheroids and patients

Background: Cancer stem cells pose significant clinical challenges in patients with colorectal cancer, as they complicate chemotherapy treatment and reduce survival rates. Investigating cancer stem cells may help elucidate the mechanisms driving therapeutic resistance and disease progression. Recent studies suggest that circular RNAs play crucial roles in tumor formation and may offer potential for early diagnosis and novel treatment strategies. However, comprehensive validations of these circular RNAs remain limited to date. The aim of this study was to elucidate the molecular roles of circular RNAs that are downregulated in colorectal cancer stem-like cell populations. Method: To explore circRNA profiles in colorectal cancer, we established three-dimensional spheroid cultures using HCT-15 and WiDr cell lines to model cancer stem cells. Bulk RNA sequencing was conducted on extracted RNA to profile circRNA and mRNA expression. Bioinformatic analyses, including profiling of transcriptomic alterations and development of interaction frameworks among circRNAs, miRNAs, and mRNAs, were performed using CircInteractome, CircBank, miRDB, and TargetScan to elucidate relationships among target genes. Results: Circular RNA sequencing revealed a predominant downregulation of circRNAs in spheroid cultures. Integration with clinical datasets comprising 788 colorectal cancer patients identified 15 circRNAs consistently downregulated in both spheroid models and metastatic tissues. Among these, hsa_circ_0039776 and hsa_circ_0067353 emerged as key tumor-suppressive candidates. These circRNAs were predicted to regulate three oncogenic miRNAs—hsa-miR-614, hsa-miR-645, and hsa-miR-4689— which were significantly upregulated in advanced-stage colorectal cancer patients. Cross-validation using CircInteractome, CircBank, miRDB, and TargetScan confirmed the circRNA–miRNA and miRNA–mRNA interactions. Further analyses identified four tumor-suppressive genes (CCL5, INSC, SLC9A1, and SLC34A1) associated with improved patient survival. Gene set enrichment analysis suggested that these regulatory networks modulate cancer stem cell phenotypes, including inhibition of differentiation, resistance to apoptosis, and enhancement of DNA damage repair pathways. These findings were consistently observed across both HCT-15 and WiDr cell lines, demonstrating reproducibility. Conclusion: This study identifies hsa_circ_0039776 and hsa_circ_0067353 as novel tumor-suppressive circRNAs exhibiting reduced expression in colorectal cancer spheroids and metastatic lesions. The constructed circRNA–miRNA–mRNA regulatory network indicates that these circRNAs modulate oncogenic miRNAs (hsa-miR-614, hsa-miR-645, hsa-miR-4689) and their downstream tumor-suppressive targets (CCL5, INSC, SLC9A1, SLC34A1), which correlate with favorable survival outcomes and influence cancer stemness. These results contribute to a deeper comprehension of the role of circRNAs in the pathogenesis of colorectal cancer and underscore their potential utility as diagnostic biomarkers and therapeutic targets.