Background/Purpose: Long non-coding (lnc-)RNAs are transcribed from DNA similar to mRNA. lncRNA are able to interact directly with DNA, RNA and proteins. Some lncRNAs contain micro (mi-)RNAs in their sequence. The miRNAs can be released by splicing leading to active miRNA molecules. lncRNA H19 includes two miRNAs 675-3p and -5p in its sequence. Adipose tissue derived adipokines are involved in inflammation processes as well as osteoarthritis (OA) development. The proinflammatory adipokine visfatin is able to alter osteogenic differentiation (OD) of pluripotent mesenchymal stem cells (MSCs). Visfatin reduces elastic fiber expression, increases matrix mineralization and proinflammatory factor production.

Methods: MSCs isolated from OA hip or knee bone (phMSCs) and commercially obtained healthy human hMSCs were differentiated towards osteoblasts stimulated with or without visfatin, resistin, leptin as well as TNF and Wnt/TGFβ1 pathway inhibitors. Supernatants were collected at days 2, 7, 9, 14 and 21 of OD, cell lysates at day 2, 7, 9, 14. Matrix mineralization assays were performed at day 21. lncRNA H19 and miRNA expression was evaluated by real-time PCR after miRNA and total RNA isolation. IL-6 was measured by ELISA.

Results: Visfatin increased matrix mineralization and IL-6 release (hMSC: p=0.03, phMSC: p=0.01) (1). lncRNA H19 was continuously upregulated in unstimulated controls during OD as well as with leptin or resistin. Stimulation with visfatin significantly decreased lncRNA H19 (d2 to d14 of OD, phMSC: p=0.01, h-MSC: p=0.04). TNF stimulation during OD did not lead to downregulation of H19 nor increased matrix mineralization. lncRNA H19 endogenous miRNA 675-5p was reduced in parallel with H19: increased during OD and downregulated by visfatin significantly (e.g. d14 p=0.02). However, H19 endogenous miRNA 675-3p was inversely regulated: downregulated during OD while visfatin attenuated this effect (e.g. d14 p=0.03). Altered Wnt-signaling or TGFβ1 pathway were not observed.

Conclusion: lncRNA H19 is upregulated during OD playing a regulatory role during osteogenesis. During OD, visfatin showed proinflammatory effects and increased matrix mineralization while reducing elastic fiber production. These effects were associated with a reduction of lncRNA H19, an effect not triggered by other adipokines or TNF. We demonstrated that the lncRNA H19 endogenous miRNA 675-5p was regulated in parallel to H19, whereas miRNA 675-3p was inversely regulated and increased continuously upon visfatin stimulation. These results indicate that miRNA 675-3p is released out of the lncRNA H19 sequence leading to H19 reduction representing an effector mechanism of visfatin. We hypothesize that this is a restrictive process in which either microRNA 675-5p or -3p is liberated from H19 and, in this setting, of miRNA 675-3p.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/lncrna-h19-and-micro-rna-675-3p-are-altered-by-visfatin-during-osteogenesis/