Abstract: By 2030, pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths worldwide. Its high mortality is driven by late diagnosis and ineffective treatments, underscoring the urgent need for the identification of novel targeted therapies. Circular RNAs (circRNAs) are a sub-group of ncRNAs that form a covalently closed continuous circular loop. CircRNAs play a significant role in regulating the transcription of oncogenes and tumor suppressor genes through miRNA sponging and RNA binding protein (RBP) interactions. Studies have shown that circRNA expression patterns can serve as prognostic markers for PDAC. However, whether these circRNAs can be targeted using less toxic treatment strategies, such as natural product derivatives, has not been explored. Here, we evaluated the impact of thymoquinone (TQ), a derivative of Nigella sativa (black seed), on PDAC-associated circRNAs.

Methods: We performed circRNA-seq profiling on MiaPaCa-2 and Panc-1 PDAC cell lines treated with TQ. The expression of the top prioritized differentially expressed circRNAs was verified by qRT-PCR using divergent primers. The biological relevance of the selected circRNAs modified by TQ was assessed using the publicly available PDAC circRNA microarray dataset (GSE79634), and pathway analysis was employed to evaluate their biological targets.

Results: TQ treatment resulted in the downregulation of PDAC gene set enrichment analysis (GSEA) identified oncogenic circRNAs, including hsa_circ_0000567, hsa_circ_0008344, hsa_circ_0058453, hsa_circ_0004405, hsa_circ_0012152, hsa_circ_0008193, hsa_circ_0001900, hsa_circ_0001495, hsa_circ_0006877, hsa_circ_0007643, hsa_circ_0002402, hsa_circ_0000707, and hsa_circ_0001558. GSEA revealed that the TQ-altered circRNAs were associated with the Wnt and Hedgehog signaling pathways. RT-PCR demonstrated that the identified circRNAs were elevated in PDAC cell lines compared to normal HPNE cells, and their expression was significantly reduced following TQ treatment (p < 0.05). Furthermore, circR-0058453 was found to bind to specific miRNAs, miR-1248 and miR-1287, which have been reported to function as tumor suppressors. These findings suggest that circR-0058453 may act as an oncogenic circRNA by sponging tumor-suppressive miRNAs, thereby promoting PDAC growth—a process that can be reversed by TQ. Notably, circR-0058453 contains 22 binding sites for AGO2, and RNA immunoprecipitation assays confirmed that its binding to AGO2 was downregulated by TQ. These results suggest that TQ exerts its inhibitory effects on cell signaling through the regulation of circRNA and RBP interactions.

Conclusions: This study is the first to demonstrate that PDAC associated circRNAs can be targeted by the non-toxic natural product thymoquinone that warrants further clinical investigation.