Design and Testing of Small Interfering RNAs (siRNAs) Targeted Against Equine TNF‐α and IL1‐β in Equine Synoviocyte Cultures – Preliminary Findings

Abstract

AimsSynovial inflammation is characterised by increases in catabolic cytokines, notably tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β); to understand their role in equine arthritis, model systems to evaluate small interfering RNAs (siRNAs) targeted against these cytokines are being developed. Specifically, we aim to investigate the change in TNF‐α and IL‐1β expression following transfection of equine synoviocytes with TNF‐α siRNA and IL‐1β siRNA. It is hypothesised that gene expression would decrease following treatment with siRNAs, and that off‐target effects would not be induced.MethodsSynovium from healthy metacarpophalangeal joints of 6 horses was digested using 0.2% trypsin and 0.2% collagenase type‐II. Synoviocytes were cultured in DMEM with 10% FCS, with and without 1 μg/ml LPS, and with and without one of 3 siRNAs at 20 nmol: generic scrambled control siRNA, TNF‐α siRNA or IL‐1β siRNA. Gene expression was analysed with RT‐qPCR for TNF‐α and IL‐1β; GAPDH was used for normalisation.ResultsThe patterns of TNF‐α gene expression in 2 experiments were similar; expression levels decreased (51%) relative to LPS stimulated controls when using TNF‐α siRNA. Efficiency of transfection with the IL‐1β siRNA was less reliable: expression levels of IL‐1β actually increased when using IL‐1β siRNA.ConclusionsThe pattern of TNF‐α gene expression supported our hypothesis but the degree of gene knockdown was disappointing. This may be due to a number of reasons; LPS concentration, siRNA transfection method, or siRNA sequence. The IL‐1β siRNA did not work in these experiments and indeed may be inducing ‘off‐target’ effects. The mixed culture of synoviocytes used was predominantly synovial fibroblasts. Further work will be carried out using peripheral blood mononuclear cells, and multiple siRNA sequences.Practical significanceTraumatic arthritis is common in Thoroughbred racehorses and is a significant cause of wastage in the industry.Ethical animal researchApproval was granted by the Liverpool University Ethics Committee. Sources of funding: Rhiannon Morgan is in receipt of a Veterinary Research Training Scholarship from the Horserace Betting Levy Board. Competing interests: None.