Thymoquinone blocks pancreatic ductal adenocarcinoma growth by altering novel circRNAs and miRNAs

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths worldwide underscoring the urgent need for the identification of novel therapeutical strategies. Circular RNAs (circRNAs) are a sub-group of small non-coding RNAs with a closed loop structure generated by back splicing of RNAs which play a significant role in the regulation of transcriptional and post-transcriptional processes of many genes. Altered regulations of circRNAs result in tumorigenesis and progression of a wide variety of tumors. To date there are no studies that have evaluated whether circRNAs could be modulated using natural agent derivatives for therapeutic benefit. The objective of this study is to identify tumor associated circRNAs and miRNAs modulated by black seed derivative Nigella sativa thymoquinone (TQ) in PDAC models. Methods: circRNA-seq, RNA pull-down, cell viability, RT-qPCR, CSC sphere formation, colony formation assays and miRNA transfection technique. TQ treatment resulted in the downregulation of PDAC-oncogenic circRNAs, along with the inhibition of cell growth, CSC self-renewal capacity, and clonogenic capacity. RT-qPCR showed that TQ suppressed the expression of 5 of the sequencing identified circRNAs (hsa_circ_0054853, hsa_circ_0012152, hsa_circ_0001495, hsa_circ_0006877, and hsa_circ_0000567) that were inherently elevated in PDAC and not in normal cells. CircR-0054853 was found to bind to specific tumor suppressor miRNAs, miR-1248 and miR-1287. The forced-expressions of these miRNAs result not only in the inhibition of cell growth but increased the sensitivity to TQ in PDAC cells as TQ treatment increased expressions of these miRNAs. These findings suggest that circR-0054853 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-0054853 contains 22 binding sites for AGO2, and RNA immunoprecipitation assays confirmed that its binding to AGO2 was downregulated by TQ. We also found that TQ significantly increased the efficacy of gemcitabine/nap-paclitaxel or pan Kras inhibitor RMC6236 in Kras mutant PDAC in vitro and animal models. The co-treatment of TQ with RMC6236 showed a remarkable combination effect on EZH2 and REK/MEK pathway in Kras mutant cells. In conclusion, for the first time we report that TQ has anti-tumor activity and exerts its inhibitory effects on tumor-associated cell signaling through the regulations of circRNAs, miRNAs and RBP interactions that warrants further pre-clinical and clinical investigations.