Investigating the roles of JUP, HMGB1, and FASN knockdown using pro-siRNA mediated regulation on TRAIL and TNF-α induced apoptosis.

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Background: Evasion of apoptosis is a fundamental hallmark of cancer. Restoring the body’s natural programmed cell death process through death ligands, such as TRAIL and TNF-α, represents a promising therapeutic strategy. However, the roles of specific genes in mediating these pathways remain complex. This study utilizes a novel, cost-effective RNA interference technology—prokaryotic siRNAs (pro-siRNAs)—to investigate the roles of Fatty Acid Synthase (FASN), Junction Plakoglobin (JUP), and High-Mobility Group Box 1 (HMGB1) in apoptotic regulation with different cancer cell lines. The study aimed to investigate whether FASN, JUP, and HMGB1 act as essential mediators of TRAIL- and TNF-α-induced apoptosis in human carcinoma cell lines. We hypothesized that silencing these genes would confer resistance to death ligands. Additionally, we sought to validate the efficacy of bacterially produced pro-siRNAs against conventional chemically synthesized siRNAs. Methods: Recombinant pro-siRNAs targeting FASN, JUP, and HMGB1 were produced. Their knockdown efficiency was evaluated using RT-qPCR. Following 48-hour transfection using Lipofectamine RNAiMAX with a final pro-siRNA concentration of 50 nM, cells were treated with varying concentrations of TRAIL or TNF-α for 24 hours. Apoptotic response was quantified via high-content screening of relative caspase activity and cell viability. Results: RT-qPCR confirmed knockdown efficiencies exceeding 80% for all target genes across all cell lines. Knockdown of JUP and HMGB1 in Caco2 cells unexpectedly conferred significant resistance to TNF-α-induced apoptosis, reducing relative caspase activity by approximately 53.6% and 53.1%, respectively, in both cases compared to siNC controls. However, in both HeLa and Huh7 cells, the silencing of these genes did not significantly alter the cell's response to the death ligands. These results demonstrate the potential of pro-siRNA technology as a cost-effective and scalable approach for targeted cancer therapy. Conclusions: The study confirms the utility of pro-siRNA technology as a scalable tool for targeted gene silencing. While JUP and HMGB1 knockdown did not universally inhibit apoptosis, their silencing provided a protective effect against TNF-α in Caco2 cells. This highlights significant cellular heterogeneity and suggests that colorectal cancer cells may utilize distinct compensatory survival mechanisms when specific apoptotic mediators are suppressed.