類風濕性關節炎是一種慢性自體免疫疾病，能引起多處週邊關節發炎，其病徵為關節內滑膜組織發炎增厚帶有白血球浸潤，大量增生的滑膜纖維母細胞聚集形成血管翳，進而侵蝕軟骨、破壞硬骨而造成關節受損。在類風濕性關節炎的滑膜纖維母細胞內長鏈非編碼核糖核酸(long non-coding RNA, lncRNA)具有調控其致病性的角色，過去的研究發現lncRNA H19在類風濕性關節炎病人的滑膜組織中有異常高表現並在其致病機轉中有潛在性之重要功能。本研究吾人探討H19參與tumor necrosis factor (TNF)調控之活化滑膜纖維母細胞的功能，並以clustered regularly interspaced short palindromic repeats interference (CRISPRi) 檢驗減少H19在滑膜纖維母細胞中的表現量對類風濕性關節的治療效果。我們發現類風濕性關節炎患者的滑膜組織、分離出的滑膜纖維母細胞及周邊單核白血球之H19表現量明顯較對照組退化性關節炎病患檢體高，而臨床上施打TNF單株抗體三週後類風濕性關節炎病人的周邊單核白血球中H19表現量下降。我們進一步證明以TNF刺激滑膜纖維母細胞使其H19及enhancer of zeste homolog 2 (EZH2)表現量增加，顯示兩者在滑膜纖維母細胞之表現可能受TNF調控。以慢病毒載體CRISPRi-H19轉殖之大鼠關節炎滑膜纖維母細胞相較對照組載體CRISPRi-GFP轉殖者EZH2、phosphorylated GSK-3β及Snail表現量均減少，其IL-6分泌量及細胞侵略能力皆下降。最後我們以慢病毒載體CRISPRi-H19在關節炎模式大鼠進行關節內注射實驗，顯示減少H19基因表現量在大鼠關節炎中可減緩其發炎。本研究結果顯示在類風濕性關節炎之滑膜纖維母細胞中H19可透過EZH2-Wnt signaling-Snail路徑來調節TNF調控之活化。

In rheumatoid arthritis (RA), an autoimmune disease affecting peripheral joints, there are hyperplastic synovial membranes infiltrated with immune cells and synovial fibroblasts (SFs). Activated SFs with inflammatory and invasive phenotypes are responsible for synovitis, cartilage erosion and joint destruction. Previous reports have shown that the long non-coding RNA (lncRNA) H19 plays an important role in the tumorigenesis and is overexpressed in RA synovial tissues (STs) with a potential implication in the pathogenesis. In this study, we explored whether H19 is involved in tumor necrosis factor (TNF)-mediated SF activation in rheumatoid joint. H19 expression levels were higher in peripheral blood mononuclear cells (PBMCs), STs, and SFs of RA patients in comparison with osteoarthritis counterparts, as determined by quantitative real-time PCR. After receiving anti-TNF injection for 3 weeks, H19 expression levels were decreased in PBMCs of RA patients. Furthermore, H19 and enhancer of zeste homolog 2 (EZH2) expression were induced in SFs in response to TNF stimulation. To further elucidate the pathogenic mechanisms of H19 in SF activation, we used lentivirus-based CRISPR interference (CRISPRi) targeting H19 (LVCRISPRi-H19). The expression levels of EZH2, phosphorylated GSK-3β, and Snail were down-regulated in LVCRISPRi-H19-transduced SF transfectants. Silencing H19 expression in SFs reduced the cell invasive capacity and IL-6 secretion. Moreover, targeting synovial H19 expression through intra-articular injection of LVCRISPRi-H19 ameliorated collagen-induced arthritis. Collectively, our results suggest that H19 regulates the activation of SF in rheumatoid joint through the EZH2-Wnt signaling-Snail pathway.

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