gms | German Medical Science

Introduction: Long non-coding (lnc-)RNA transcribed from DNA similar to mRNA are able to interact directly with DNA, RNA and proteins. Several lncRNAs contain micro (mi-)RNAs that can be released by splicing. lncRNA H19 includes two miRNAs 675-3p and -5p. Adipose tissue derived adipokines are involved in inflammatory processes and osteoarthritis (OA) development. The proinflammatory adipokine visfatin alters osteogenic differentiation (OD) of pluripotent mesenchymal stem cells (MSCs), reduces elastic fiber expression, increases matrix mineralization and release of cytokines and chemokines [1]. Therefore, we evaluated the effect of visfatin on lncRNA H19 in MSCs during OD to explore the kinetics of the effect of visfatin on H19 regulation during OD and elucidate the respective downstream mechanisms.

Methods: MSCs isolated from OA knee/hip bone (phMSC) and commercially obtained healthy (h-)MSCs were differentiated towards osteoblasts with/without visfatin/resistin/leptin/TNF and Wnt/TGFβ1 pathway inhibitors. Supernatants were collected at days 2/7/9/14/21 of OD, cell lysates at day 2/7/9/14 and matrix-mineralization assays at day 21. H19 and miRNA expression was evaluated by realtime-PCR after mi-/RNA isolation. IL-6 was analyzed by ELISA.

Results: H19 was continuously upregulated in unstimulated controls during OD, but also when stimulated with resistin/leptin. Stimulation with visfatin significantly decreased H19 (d2-d14 of OD, hip-phMSCs: p=0.0097, knee-phMSCs: p=0.0075, h-MSC: p=0.044). Visfatin increased matrix-mineralization and IL-6 production (hMSC: p=0.03, phMSC: p=0.013)(1). TNF stimulation during OD did not lead to downregulation of H19 nor increased matrix mineralization. H19-endogenous miR675-5p was increased in parallel with H19 during control OD and significantly downregulated by visfatin (e.g. day 14 p=0.015). miR675-3p was downregulated during control OD while visfatin attenuated this effect (e.g. d14 p=0.025). Altered Wnt-signaling and involvement of the TGFβ1 pathway was not observed.

Conclusion: H19 is upregulated during OD and may play a regulatory role in osteogenesis. Visfatin showed pro-inflammatory effects during OD, increased matrix-mineralization while reducing elastic fiber production(1). These effects were associated with H19 downregulation, a visfatin-effect not triggered by other adipokines or TNF. We demonstrated miR675-5p to be downregulated in parallel to H19, whereas endogenous miR675-3p was inversely upregulated. These results indicate that miRNA 675-3p can be spliced out of H19 leading to H19 reduction representing a potential effector mechanism of visfatin.

Disclosures: none