Background/Purpose: Aberrantly higher expression of long non-coding RNAs (lncRNAs) in synovial fibroblasts (SFs) plays pathogenic roles in rheumatoid joint. Studying the effects on knocking down lncRNAs expression in arthritis models would contribute to the development of lncRNAs-related therapeutics in rheumatoid arthritis (RA). We examined whether intra-articularly (i.a.) delivering the lentivirus (LV)-based CRISPR interference (CRISPRi) targeting H19, an oncofetal lncRNA involved in tumor metastasis, in SFs can ameliorate experimental arthritis.

Methods: H19 expression levels were examined by quantitative real-time PCR (qRT-PCR) in mononuclear cells (MNCs) from RA before and after receiving a TNF blockade therapy and osteoarthritis (OA) patients. Synovial tissues were from arthritis patients and an experimental model, collagen-induced arthritis (CIA). SFs were purified from patients, and normal human SFs were obtained commercially. Single guide RNA oligonucleotides that directing Cas9 to target sites were designed according to available algorisms. A 1.9 kb stuffer was removed from LV plasmid pAll-dCAS9-KRAB.pPuro for cloning, and created single guide RNA vectors were transiently transfected into 293T cells to obtain recombinant LV vectors. SFs were transduced with LVCRISPRi-H19 under polybrene, followed by selection with puromycin incubation to produce stable H19-silenced transfectants. Cell lysates were subjected to immunoblot with anti-EZH2, anti-pGSK-3β (a Wnt signaling) and anti-Snail. Cell invasion was assayed by Transwells with membrane coated with Matrigel. Spernatant IL-6 was quantified by ELISA. Arthritis indexes and histological scores were evaluated in CIA joints receiving LVCRISPRi-H19 or LVCRISPRi-GFP (negative control) injection, and synovial H19 expression was examined by qRT-PCR.

Results: Synovial tissues, SFs and MNCs from RA had higher H19 expression as compared with OA patients. RA MNCs had lower H19 expression after a TNF blockade injection. H19 and EZH2 levels were up-regulated in normal SFs in the presence of TNF in vitro. Lower EZH2, pGSK-3β and Snail expression was found in H19-silenced SF transfectants. LVCRISPRi-H19-injected CIA joints with lower synovial H19 expression had reduced arthritis indexes and histological scores.

Conclusion: Our results demonstrate that a TNF-mediated lncRNA pathway with H19-EZH2-Wnt signaling-Snail axis might exist in RASFs, and i.a. delivering CRISPRi targeting lncRNA H19 in SFs could ameliorates experimental arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/intra-articularly-delivering-lentivirus-based-crispr-interference-targeting-long-non-coding-rna-h19-in-synovial-fibroblasts-ameliorates-experimental-arthritis/