Investigating the Roles of JUP, HMGB1, and FASN Knockdown Using pro-siRNA Mediated Regulation on TRAIL and TNF- α Induced Apoptosis in Liver and Colon Carcinoma Cell Lines.

Abstract

The development of targeted cancer therapies is a critical area of research, as conventional treatments often have severe side effects and limited efficacy against drug-resistant tumors. One promising strategy involves reactivating the body’s natural process of programmed cell death called apoptosis. Often, cancer cells suppress these apoptotic pathways to survive, therefore, it represents a promising target for therapeutic intervention. This study aimed to investigate the effectiveness of a novel pro-siRNA technology for silencing key anti-apoptotic genes in different human carcinoma cell lines. We evaluated the effects of silencing JUP, HMGB1, and FASN in HeLa, Huh7, and Caco2 cells. Using RT-qPCR, we confirmed the knockdown of the target genes at the mRNA level. Additionally, chemically synthesized siRNAs were employed as comparators to validate the efficacy of the pro-siRNA constructs. Knockdown of JUP and HMGB1 in Caco2 cells unexpectedly conferred significant resistance to TNF-α-induced apoptosis, reducing relative caspase activity by approximately by 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, while also highlighting the cellular heterogeneity and complex survival mechanisms that must be considered in future research and clinical translation.