RNA Interference-Mediated Suppression of RAGE

The receptor for advanced glycation end products (RAGE/AGER) is an important mediator of inflammatory signaling and has been implicated in chronic low-grade inflammation associated with aging. Chronic low-grade inflammation (“inflammaging”) is a characteristic feature of biological aging, providing a rationale for investigating molecular regulators of inflammatory signaling such as RAGE. This study aimed to develop and validate short hairpin RNA (shRNA) constructs suppressing AGER/RAGE in human cell models. Five shRNA constructs targeting human AGER were designed, cloned into pLSL-puro, and verified by Sanger sequencing. Lentiviral vectors were produced in HEK293T cells and used to establish stable HEK293T, 786-O, and human umbilical vein endothelial cell (HUVEC) populations with AGER knockdown. AGER/RAGE expression was assessed by RT-qPCR, Western blotting, and flow cytometry. Silencing efficiency varied with the construct and the cellular model. sh1 produced the strongest suppression of AGER, exceeding 70% in selected models, whereas sh2 was effective in HEK293T and 786-O cells. Reduced RAGE expression was confirmed at the mRNA, protein, and cell-surface levels, identifying sh1 and sh2 as lead candidates. An AGER-focused workflow for lentiviral shRNA construct screening and experimental validation was established, providing a reproducible approach for generating stable cell models with reduced AGER/RAGE expression. Further work is required to confirm isoform specificity, assess off-target effects, and define the functional consequences of RAGE suppression.