¹Justus-Liebig-University, Campus Kerckhoff, Rheumatology and Clinical Immunology, Bad Nauheim
²University Hospital of Giessen Marburg, Dept. of Trauma, Hand and Reconstructive Surgery, Giessen
³Agaplesion Markus Hospital, Department of Orthopaedics and Trauma Surgery, Frankfurt am Main

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Introduction: Long-non-coding RNAs (lncRNAs) play key roles in disease pathophysiology. The lncRNA H19 is implicated in rheumatoid arthritis (RA) pathogenesis, particularly under inflammatory conditions, where its expression is elevated in RA synovial fibroblasts (RASF). H19 serves as a precursor for miR-675-3p/-5p, with miR-675-5p regulating H19 via a negative feedback loop. We investigated the expression of H19 and miR-675-3p/-5p under inflammatory conditions in RASF and the effects of H19 overexpression/knockdown on proliferation, migration, and miR-675 expression.

Methods: RASF were stimulated with IL-1β, TNF-α, IFN-γ, LPS, or visfatin for 17h. H19 expression was analyzed via real-time PCR, and miR-675-3p/-5p was quantified using TaqMan^® Small RNA assays. IL-6 levels were measured by ELISA. H19 overexpression and knockdown were achieved using plasmids and siRNA transfection, respectively. Proliferation was assessed via BrdU assays, and migration was evaluated using Boyden chamber assays.

Results: IL-1β, TNF-α, LPS, and visfatin significantly increased IL-6 secretion (p<0.05), while IFN-γ had no effect. However, H19 expression remained unchanged under stimulation under non-starving conditions. IL-1β and TNF-α did not alter miR-675 levels, but IFN-γ and LPS upregulated both, miR-675-3p (3.8- and 4.2-fold) and miR-675-5p (3.5- and 1.5-fold). H19 overexpression peaked at 24h (2244-fold), increasing IL-6 levels (1,035 pg/mL vs. 773 pg/mL). Early overexpression (2–6 h) led to H19 decline with miR upregulation, peaking at 24 h. In the late phase (24–72 h), both H19 and miRs declined. H19 overexpression significantly reduced proliferation (p<0.05) and migration (p<0.001), while knockdown reduced miR-675 expression but had no effect on cellular functions.

Conclusion: Under non-starved conditions, pro-inflammatory cytokines did not significantly alter H19 or miR-675 expression in RASF suggesting cell stress required for H19 alteration in RASF. H19 overexpression led to synchronized miR-675 upregulation and established a negative feedback loop. Overexpression suppressed RASF proliferation and migration, while knockdown reduced miR-675 expression without affecting cellular functions suggesting that stress-induced H19 induction in RASF leads to altered cellular function which is not visible under non-inflammatory conditions.

Disclosures: Nothing to disclose.