Identification of microRNA-181a-5p and microRNA-4454 as mediators of facet cartilage degeneration
Akihiro Nakamura, Y. Raja Rampersaud, Anirudh Sharma, Stephen J. Lewis, Brian Wu, Poulami Datta, Kala Sundararajan, Helal Endisha, Evgeny Rossomacha, Jason S. Rockel, Igor Jurisica, Mohit Kapoor
Akihiro Nakamura, Y. Raja Rampersaud, Anirudh Sharma, Stephen J. Lewis, Brian Wu, Poulami Datta, Kala Sundararajan, Helal Endisha, Evgeny Rossomacha, Jason S. Rockel, Igor Jurisica, Mohit Kapoor
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Research Article Cell biology

Identification of microRNA-181a-5p and microRNA-4454 as mediators of facet cartilage degeneration

Abstract

Osteoarthritis (OA) of spine (facet joints [FJs]) is one of the major causes of severe low back pain and disability worldwide. The degeneration of facet cartilage is a hallmark of FJ OA. However, endogenous mechanisms that initiate degeneration of facet cartilage are unknown, and there are no disease-modifying therapies to stop FJ OA. In this study, we have identified microRNAs (small noncoding RNAs) as mediators of FJ cartilage degeneration. We first established a cohort of patients with varying degrees of facet cartilage degeneration (control group: normal or mild facet cartilage degeneration; FJ OA group: moderate to severe facet cartilage degeneration) and then screened 2,100 miRNAs and identified 2 miRNAs (miR-181a-5p and miR-4454) that were significantly elevated in FJ OA cartilage compared with control facet cartilage. We further explored their role, function, and signaling mechanisms using computational, in vitro functional, and in vivo studies. We specifically indicate that miR-181a-5p and miR-4454 are involved in promoting inflammatory, catabolic, and cell death activity in FJ chondrocytes. This is the first report to our knowledge that identifies miR-181a-5p and miR-4454 as mediators of cartilage degeneration in FJs and potential therapeutic targets for stopping cartilage degeneration.

Authors

Akihiro Nakamura, Y. Raja Rampersaud, Anirudh Sharma, Stephen J. Lewis, Brian Wu, Poulami Datta, Kala Sundararajan, Helal Endisha, Evgeny Rossomacha, Jason S. Rockel, Igor Jurisica, Mohit Kapoor

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Figure 3

Effect of miR-181a-5p and miR-4454 mimics or inhibitors on the expression of catabolic, inflammatory, cell death, and matrix markers in facet joint osteoarthritis chondrocytes.

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Effect of miR-181a-5p and miR-4454 mimics or inhibitors on the expressio...
(A) Real-time PCR (RT-PCR) analysis of the expression of major cartilage catabolic (MMP13) and inflammatory (TNFA, IL6, and MCP1) markers and the cartilage matrix molecule COL2A1 in facet joint osteoarthritis (FJ OA) chondrocytes treated with miR-181a-5p or miR-4454 mimic compared with control mimic (n = 6/treatment). (B) PARP p85 expression relative to β-actin expression by Western blotting in FJ OA chondrocytes treated with miR-181a-5p or miR-4454 mimic compared with control mimic. Representative blot from n = 4 separate blots. Full uncut blots are shown in the supplemental figure. (A and B) *P < 0.05 between control and miR-181a-5p or miR-4454 mimics, as determined by 2-tailed Student’s t test. (C) RT-PCR analysis of miR-181a-5p and miR-4454 in FJ OA chondrocytes treated with (+) or without (–) IL-1β (n = 7/treatment). *P < 0.05, **P < 0.01, as determined by a 2-tailed Student’s t test. (D) RT-PCR analysis of catabolic, inflammatory, and cell death markers and COL2A1 in FJ chondrocytes treated with or without IL-1β and miR-181a-5p, miR-4454, or control inhibitors (n = 7/treatment). (E) Immunoblot analysis of PARP p85 protein levels relative to β-actin in FJ OA chondrocytes treated with miR-181a-5p and miR-4454 inhibitors compared with control inhibitor in the presence or absence of IL-1β. Representative blot from n = 4 separate blots. Full uncut blots are shown in the supplemental figure. (D and E) Differences in the levels of expression among miR-181a-5p, miR-4454, and control inhibitors with or without IL-1β treatment were determined 1-way analysis of variance followed by Tukey’s post-hoc tests.*P < 0.05, **P < 0.01, control inhibitor without IL-1β treatment vs. control inhibitor with IL-1β treatment. †P < 0.05, ††P < 0.01, control inhibitor treatment vs. miR-181a-5p or miR-4454 inhibitor treatment in the presence of IL-1β, respectively. All other comparisons were not significantly different (P > 0.05). For data presented as box-and-whiskers plots, horizontal lines and cross marks indicate the medians and the means, boxes indicate 25th to 75th percentiles, and whiskers indicate minimum and maximum values of the data set.