Novel MicroRNAs as Biomarkers and Therapeutics for Early-Stage Knee Osteoarthritis

Document Type

Conference Proceeding

Publication Date

4-1-2026

Publication Title

Osteoarthritis Cartilage

Keywords

alizarin red s, argonaute 2 protein, biological marker, immunoglobulin G, microRNA, RNA induced silencing complex, small untranslated RNA, aged, articular cartilage, biogenesis, bioinformatics, cartilage degeneration, cell differentiation, computer model, conference abstract, controlled study, etiology, explant, fat pad, gene expression, genetic transfection, human, human tissue, in vitro study, infrapatellar fat pad, Kellgren-Lawrence grade, knee, knee joint, knee osteoarthritis, MC3T3-E1 cell line, meniscectomy, mineralization, mouse, osteoarthritis, osteoblast, osteophyte, real time polymerase chain reaction, receiver operating characteristic, RNA immunoprecipitation, secondary prevention, sham procedure, subchondral bone, synovium

Abstract

Purpose (the aim of the study): There are currently no molecular biomarkers for the detection of early-stage knee osteoarthritis (OA) and no approved therapeutics to mitigate knee OA progression. MicroRNAs (miRNAs) – small, non-coding RNAs – have the potential to meet both needs. Found in biofluids and tissues, miRNAs have emerged as promising biomarkers and therapeutic targets. First transcribed as primary miRNAs (pri-miRNAs), miRNAs are processed into mature forms, then loaded into Argonaute 2 (AGO2)-containing RNA-induced silencing complexes (RISCs) to repress target gene expression. While established miRNAs (i.e., those cataloged in databases) have been explored in OA, novel miRNAs (i.e., those predicted using bioinformatics) have not been characterized. Novel miRNAs are particularly promising as biomarkers and therapeutics given their potential disease-specific associations. In the first study to profile circulating novel miRNAs in OA, Ali et al. reported four novel miRNAs (novel_miRNA_1 to _4) in plasma from individuals with early- versus late-stage knee OA. Here, we aim to comprehensively characterize these novel miRNAs and assess their roles as biomarkers and therapeutics for early-stage knee OA. Methods: In independent cohorts, we used real-time PCR to measure plasma levels of the four novel miRNAs in non-OA controls, early-stage knee OA, and late-stage knee OA. Non-OA controls included individuals with no symptomatic or radiographic evidence of knee OA. Symptomatic patients with radiographic Kellgren-Lawrence (KL) grades 0 and 1 were considered early-stage knee OA, and those with KL grades 3 and 4 were considered late-stage knee OA, as determined by an orthopaedic surgeon. To assess miRNA biogenesis by knee OA tissues, we quantified pri-miRNA and mature miRNA levels in early- and late-stage knee OA subchondral bone (“bone”), infrapatellar fat pad (“fat pad”), synovium, and articular cartilage. To assess therapeutic effects, we performed partial medial meniscectomy or sham surgery in 12-week-old mice, followed by weekly intravenous delivery of a custom-designed novel miRNA mimic or control, beginning at either one week (“early treatment”) or four weeks (“late treatment”) post-surgery. At 20 weeks of age, animals were sacrificed, and knee joint pathology was evaluated histologically. To assess canonical miRNA function, we performed AGO2-RNA immunoprecipitation in primary human tissue. To assess molecular mechanisms, direct target genes were predicted using in silico approaches, then measured in vitro in human knee OA tissue transfected with novel miRNA mimic or control. Finally, we differentiated murine pre-osteoblast cells (MC3T3-E1) and assessed mineralization with von Kossa and Alizarin Red S staining after transfection with novel miRNA mimic or control. Results: Of the four novel miRNAs, only novel_miRNA_2 was significantly elevated in plasma from early-stage knee OA compared to both non-OA controls and late-stage knee OA (Figure 1). Area under the receiver operating characteristic curve analyses showed circulating novel_miRNA_2 had excellent and good accuracy in distinguishing early-stage knee OA from non-OA controls and late-stage knee OA, respectively. Across knee OA tissues, both pri-novel_miRNA_2 and mature novel_miRNA_2 were significantly elevated in early- versus late-stage knee OA bone. In our preclinical model of knee OA, novel_miRNA_2 mimic significantly reduced osteophyte burden and cartilage degradation when delivery began in early, but not late, stages of disease. In human knee OA bone, novel_miRNA_2 was enriched in AGO2 fractions versus IgG fractions, suggesting it can be loaded into RISCs. In silico analyses predicted 13 direct target genes of novel_miRNA_2 based on bone-related involvement in OA pathogenesis, including RUNX2. Following treatment of human knee OA bone explants with novel_miRNA_2 mimic, we found reduced expression of RUNX2. In murine pre-osteoblast cells treated with novel_miRNA_2 mimic, we found reduced mineral deposition. Conclusions: Our findings sug est novel_miRNA_2 is a promising candidate biomarker and therapeutic for early-stage knee OA. We show novel_miRNA_2 functions as a canonical miRNA that regulates bone-related processes and protects against knee OA in early stages. With further research, this novel miRNA could support precision medicine approaches where its circulating levels are used to identify patients who would benefit from therapeutic supplementation to attenuate knee OA. [Formula presented]

Volume

34

First Page

S80

Last Page

S81

Find It @ Sladen

Share

COinS