The promising miRNAs, Let-7b-5p and miR-24-3p, have the potential to serve as therapeutic agents targeting pancreatic cancer stem cells.[RNA-Seq]
Background Due to its aggressive nature and poor prognosis, the development of new therapeutic strategies for pancreatic cancer is urgently needed. A promising approach involves targeting cCancer Sstem Ccells (CSCs), which are crucial in tumor growth, resistance to radiation and chemotherapy, and recurrence. Recent research has emphasized the significant role of microRNAs (miRNAs) in regulating critical aspects of CSC biology, including self-renewal, drug resistance, tumorigenicity, expression of core pluripotency factors and the modulation of genes involved in key signaling pathways. Therefore, a better understanding of how miRNAs control gene expression in Ppancreatic Ccancer Sstem Ccells (PCSCs) may help identify novel miRNAs that could serve as potential therapeutic targets for these cells. Methods /Results To identify miRNAs that are exclusive to PCSCs, the pancreosphere technique was employed to enrich them. Fluorescence-aActivated Ccell Ssorting (FACS) was used in conjunction with the surface markers CD44, CD24, and CD133 to isolate cells positive for these markers (PCSC+) and those negative for them (PCSC-). 31 Differentially Eexpressed miRNAs (DEmiRNAs) were identified using microRNA microarrays (V4.0) by hybridizing RNA from the PCSC+ and PCSC- populations. KEGG analysis revealed that 10 of the 31 downregulated DEmiRNAs are involved in signaling pathways regulating the pluripotency of PCSCs, such as Wnt/ß-catenin, TGFß, MAPK, and PI3K/AKT. These miRNAs target various mRNAs encoding ligands, receptors, and downstream effector molecules of these pathways, with results further corroborated by RT-qPCR  and Western blot analysis. Transfection of mimics of Let-7b-5p and miR-24-3p into PCSC+ cells resulted in their overexpression, which reduced the activity of key signaling pathways and pluripotency factors, inducing differentiation into exocrine and endocrine phenotypes, as corroborated by, RT-qPCR, Western blot and RNA-seq data. FACS, gemcitabine sensitivity, colony formation assays, and in vivo tumorigenicity experiments showed that the overexpression of each of these miRNAs increased the sensitivity of PCTC+ cells to gemcitabine, decreased their numbers, resulted in the formation of smaller spheres, and development of smaller tumors in vivo. Conclusion Our findings highlight the potential of miRNAs Let-7b-5p and miR-24-3p as therapeutic targets. Their ability to regulate key CSC pathways suggests that reintroducing tumor-suppressive miRNAs could reprogram PCSCs and improve patient outcome. Overall design: RNA-seq profiling of PCSC+ cells transfected with Let-7b-5p or miR-24-3p mimics.