Background/Purpose: Autophagy gets rids of unwanted proteins or organelle keeping cells to stay functional upon encountering adverse stresses. While non-coding microRNAs interfere with mRNA targets engaging in tissue metabolism, their actions to chondrocyte autophagy during osteoarthritis (OA) pathogenesis remain elusive. This study is aimed to utilize human knee OA specimens, and chondrocyte-specific microRNA-128 (miR-128) knockout mice to verify the role of miR-128a signaling in chondrocyte autophagy and OA.

Methods: OA articular were harvested from 28 patients with end-stage knee OA who required total knee arthroplasty, non-OA cartilage tissues were collected from 17 participants with a femoral neck fracture. Articular integrity in chondrocyte-specific miR-128 knockout mice (Col2α1-cre miR-128^fl/fl) and wild-type mice upon destabilized medial meniscus (DMM) surgery or intra-articular injection of collagenase were characterized using equilibrium partitioning ionic contrast-μCT imaging, histomorphometry, and immunostaining. Autophagosome, autophagic marker expression, and apoptosis of chondrocytes were quantified using RT-PCR, immunofluorescence, and annexin V-probed flow cytometry.

Results: Human OA cartilage showed 60-72% decreases in autophagic markers Atg4, Atg12, p62, and Beclin expression, and weak autophagosome component LC3 immunostaining along with 2.3-fold increases in miR-128 expression. In vitro, forced miR-128 expression significantly inhibited autophagic program, like LC3-II conversion, Atg12 expression, and fluorescent autophagic puncta formation, speeding up apoptosis and cartilage matrix underproduction of chondrocytes. Silencing miR-128 shielded them off the IL-1β-induced inhibition of survival, autophagy, and chondrogenesis. Of interest, chondrocyte-specific miR-128 knockout mice showed abundant cartilage development within joints, like spacious cartilage morphology, dense matrix synthesis, and intensive chondrocyte proliferation along with strong autophagic reactions. More strikingly, these effects significantly compromised the severity of DMM- or collagenase-mediated cartilage breakdown, synovitis, and osteophyte deposition in affected joints improving walking patterns, body axis, and mobility. Mechanistically, miR-128 hindered chondrocyte autophagy through targeting 3’-untranslated region of Atg12 impeding its mRNA expression as evident from luciferase reporter assays. Epigenetic pathway histone methyltransferase EZH2-mediated trimethyl lysine 27 of histone 3 signaling reciprocally controlled miR-128 transcription in inflamed chondrocytes.

Conclusion: Increased miR-128 signaling correlates with aberrant chondrocyte autophagy in human knee OA. miR-128 is detrimental to autophagy causing apoptosis and matrix loss in chondrocytes. miR-128 deletion promotes cartilage development and wards off excessive articular damage slowing down OA progression. Robust analyses shed light on the inhibitory actions of miR-128 to chondrocyte function and convey a perspective of miR-128 signaling blockade strategy beneficial for cartilage anabolism.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microrna-128-impairs-cartilage-integrity-and-deteriorates-osteoarthritis-pathogenesis-through-deregulating-chondrocyte-autophagy/