Inhibition of Tubulin β-chain May Play a Regulatory Role in the Development of Rheumatoid Arthritis

Abstract

Pathological characteristics of Rheumatoid arthritis (RA), a systemic autoimmune disease, includes abnormal proliferation of synovial fibroblasts and increased angiogenesis.The study assessed the effect of tubulin β-chain gene (TBB) gene knockdown on the behaviour of rheumatoid arthritis (RA) fibroblast-like synoviocytes (SFs), which is significantly affected by the synthesis of tubulin. Cultured SFs were isolated from the mixed knee-joint synovial tissue of five RA patients and treated with siRNA targeting TBB. Knockdown efficiency and expression of relevant genes were detected by RT-PCR following 24 h and 36 h of transfection. MTT, Transwell, and wound scratch assays and flow cytometric analysis were used to assess cell proliferation and invasive and migratory capacity following TBB knockdown. The expression of TBB was detected after inhibition of the ERK, STAT3 and NF-kB pathways. The expression of IL-17, TNF-α, IL-1α, IL1-β, and IL-6 was quantified following TBB knockdown. The proliferation, invasion, and migration of RASFs were significantly decreased following TBB knockdown. ELISA showed a significant decrease in interleukin 1β (IL-1β) secretion and insignificantly reduced secretion of TNF-α, IL-17, and IL-1α after TBB knockdown. The expression of TBB in RASFs decreased with the increasing concentration of a STAT3 pathway inhibitor while TBB expression increased after down regulation of the ERK pathway. These results support a protective role for TBB knockdown in suppressing RASFs, laying a foundation for further studies on the pathogenic mechanisms of RA.

Zusammenfassung

Zu den pathologischen Charakteristika der rheumatoiden Arthritis (RA), einer systemischen Autoimmunerkrankung, zählen die abnorme Proliferation synovialer Fibroblasten sowie eine verstärkte Angiogenese. In dieser Studie wurde untersucht, wie sich ein Gen-Knockdown beim Tubulin-β-Ketten(TBB)-Gen auf das Verhalten der an der rheumatoiden Arthritis (RA) beteiligten fibroblastenähnlichen Synoviozyten (SF) auswirkt, welches signifikant von der Tubulinsynthese beeinflusst wird. Bei fünf RA-Patienten wurden kultivierte SF aus dem gemischten Synovialgewebe des Kniegelenks isoliert und mit gegen TBB gerichteten siRNA behandelt. Nach 24- und 36-stündiger Transfektion wurden mittels RT-PCR die Effizienz des Knockdowns und die Expression der relevanten Gene bestimmt. Mithilfe von MTT-, Transwell- und Wound-Scratch-Assays sowie einer durchflusszytometrischen Analyse wurden die Zellproliferation und die Migrationskapazität nach dem TBB-Knockdown beurteilt. Außerdem wurden die TBB-Expression nach Inhibition der ERK-, STAT3- und NF-kB-Signalwege ermittelt und die Expression von IL-17, TNF-α, IL-1α, IL1-β und IL-6 nach dem TBB-Knockdown quantifiziert. Die Proliferation, Invasion und Migration von RASF waren nach dem TBB-Knockdown signifikant reduziert. Im ELISA zeigten sich eine signifikante Reduktion der Interleukin-1β(IL-1β)-Sekretion und eine nicht signifikant reduzierte Sekretion von TNF-α, IL-17 und IL-1α nach dem TBB-Knockdown. Die TBB-Expression in RASF nahm mit zunehmender Konzentration eines Inhibitors des STAT3-Signalweges ab, während die TBB-Expression nach Downregulation des ERK-Signalweges zunahm. Diese Ergebnisse sprechen dafür, dass ein TBB-Knockdown eine protektive Rolle bei der Suppression von RASF einnimmt, und bilden daher eine Grundlage für weitere Studien über die pathogenen Mechanismen bei RA.

Key words

tubulin β-chain - siRNA - rheumatoid arthritis - fibroblast-like synoviocytes

Schlüsselwörter

Tubulin-β-Kette - rheumatoide Arthritis - fibroblastenahnliche Synoviozyten - siRNA

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