Engineering of siRNA molecules to silence SphK1 mRNA for therapeutic intervention in triple-negative breast cancer:...

Triple-negative breast cancer (TNBC) is an aggressive and therapeutically challenging subtype of breast cancer lacking estrogen, progesterone, and HER2 receptors. Sphingosine kinase 1 (SphK1), a key enzyme in the sphingosine-1-phosphate (S1P) signaling pathway, has been implicated in TNBC progression, metastasis, and chemoresistance. In this study, we employed a comprehensive computational and experimental approach to design and validate small interfering RNA (siRNA) molecules targeting SphK1 for therapeutic intervention. A panel of siRNAs was designed using multiple bioinformatic algorithms and evaluated through secondary structure prediction, off-target screening, and molecular docking against both SphK1 mRNA and the human Argonaute 2 (AGO2) protein. Molecular dynamics simulations confirmed the structural stability and functional compatibility of the top candidate, g6 siRNA, within the AGO2 binding pocket. Experimental validation using Lipofectamine-2000 mediated transfection in MDA-MB-231 TNBC cells demonstrated that g6 siRNA achieved approximately 75-80% reduction in SphK1 mRNA and protein expression, leading to marked inhibition of cell proliferation, migration, and colony formation, and a significant increase in apoptosis. These findings confirm the predictive reliability of our in-silico workflow and establish g6 siRNA as a potent candidate for targeted SphK1 silencing in TNBC. The study further highlights the translational potential of combining g6 siRNA with current therapeutic regimens, including PARP inhibitors, immune checkpoint inhibitors, or chemotherapeutic agents, to improve treatment efficacy and overcome drug resistance in TNBC.