Abstract
Long non-coding RNAs (lncRNAs) play pivotal roles in diseases such as osteoarthritis (OA). However, knowledge of the biological roles of lncRNAs is limited in OA. We aimed to explore the biological function and molecular mechanism of HOTTIP in chondrogenesis and cartilage degradation. We used the human mesenchymal stem cell (hMSC) model of chondrogenesis, in parallel with, tissue biopsies from normal and OA cartilage to detect HOTTIP, CCL3, and miR-455-3p expression in vitro. Biological interactions between HOTTIP and miR-455-3p were determined by RNA silencing and overexpression in vitro. We evaluated the effect of HOTTIP on chondrogenesis and degeneration, and its regulation of miR-455-3p via competing endogenous RNA (ceRNA). Our in vitro ceRNA findings were further confirmed within animal models in vivo. Mechanisms of ceRNAs were determined by bioinformatic analysis, a luciferase reporter system, RNA pull-down, and RNA immunoprecipitation (RIP) assays. We found reduced miR-455-3p expression and significantly upregulated lncRNA HOTTIP and CCL3 expression in OA cartilage tissues and chondrocytes. The expression of HOTTIP and CCL3 was increased in chondrocytes treated with interleukin-1β (IL-1β) in vitro. Knockdown of HOTTIP promoted cartilage-specific gene expression and suppressed CCL3. Conversely, HOTTIP overexpression reduced cartilage-specific genes and increased CCL3. Notably, HOTTIP negatively regulated miR-455-3p and increased CCL3 levels in human primary chondrocytes. Mechanistic investigations indicated that HOTTIP functioned as ceRNA for miR-455-3p enhanced CCL3 expression. Taken together, the ceRNA regulatory network of HOTTIP/miR-455-3p/CCL3 plays a critical role in OA pathogenesis and suggests HOTTIP is a potential target in OA therapy.
Highlights
Osteoarthritis (OA) is the most widespread chronic joint disease, characterized by progressive destruction of cartilage integrity, thickening of subchondral bone, osteophyte formation, and joint-space narrowing, rendering it a leading cause of disability (Goldring and Goldring, 2007)
Inverse expression patterns in HOTTIP, and CCL3 were found during the chondrogenic differentiation of human mesenchymal stem cell (hMSC) at 14–35 days (Figures 1B,C), suggesting that miR-455-3p may have an effect on HOTTIP and CCL3 expression
It is important to note that while moderate to high levels of miR-455-3p expression were detected in proliferating chondrocytes, little to no miR455-3p expression was observed in hypertrophic chondrocytes (Figures 1D,H)
Summary
Osteoarthritis (OA) is the most widespread chronic joint disease, characterized by progressive destruction of cartilage integrity, thickening of subchondral bone, osteophyte formation, and joint-space narrowing, rendering it a leading cause of disability (Goldring and Goldring, 2007). Chemokines are tiny chemotactic cytokines that play vital roles in inflammatory diseases such as rheumatoid arthritis (RA) and OA (Gerard and Rollins, 2001; Haringman et al, 2004). Proinflammatory factors derived from the synovium and chondrocytes, such as TNF-α and IL-1β, stimulate chemokine expression in the initial and developmental progression of OA (Gerard and Rollins, 2001). Previous studies revealed that IL-1β-dependent induction of CCL3 expression in primary human chondrocytes (PHCs) promotes cartilage degradation (Sandell et al, 2008; Zhang et al, 2010) and that plasma CCL3 could be a potential serum biomarker in knee degeneration (Zhao et al, 2015). Further research has demonstrated the essential role of CCL3 in the development of joint arthritis in CCL3-null mice (Chintalacharuvu et al, 2005)
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