STUDY OF THE EFFECT OF SMALL INTERFERING RNAs ON THE EXPRESSION OF IFN-γ, IL-4, TLR4, AND TLR7 GENES IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF PATIENTS WITH RHEUMATOID ARTHRITIS

Aim . To determine the effect of small interfering RNAs on the expression of immune response genes in peripheral blood mononuclear cells of patients with rheumatoid arthritis. Materials and methods. Peripheral blood mononuclear cells from patients with rheumatoid arthritis were isolated by density gradient centrifugation. Small interfering RNAs were introduced into peripheral blood mononuclear cells via transfection using commercial reagents (Lipofectamine). Gene expression was evaluated by real-time PCR (ΔΔCt, log₂ Fold Change) 48 hours after gene knockdown. GAPDH expression was used as an endogenous control. Statistical analysis included the nonparametric Mann – Whitney U test (p = 0.05). Results and discussion. Transfection of peripheral blood mononuclear cells with specific small interfering RNAs targeting IFN -γ , IL-4, TLR4, and TLR7 resulted in 43,9% decrease in TLR4 expression (p=0.0286) and 38.4% decrease in TLR7 expression (p=0.0211). Conclusion . The study confirmed the relevance of peripheral blood mononuclear cells using as an in vitro model for investigating molecular mechanisms, demonstrating their ability to reproduce features of the immune response. Successful transfection of target small interfering RNAs against key genes showed high precision and efficiency of the chosen method and led to a significant reduction in gene expression. Particular attention should be paid to the identified ability of small interfering RNAs to effectively suppress TLR4 and TLR7 expression, which demonstrates both the functionality of the small interfering RNAs and the susceptibility of these targets to silencing. Post-transcriptional reduction of mRNA levels is of fundamental importance, indicating that toll-like receptors are promising candidates for molecular intervention.