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Updated: May 5, 2026
Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness
Published on: March 18, 2022
NFAT3 and TGF-β/SMAD3 regulate the expression of miR-140 in osteoarthritis
Introduction:
MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.
Methods:
Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.
Results:
In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.
Conclusions:
This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.
Insights
Researchers uncovered a novel regulatory mechanism for microRNA-140 (miR-140) in osteoarthritis (OA) chondrocytes, independent of the WWP2 gene. This finding reveals new roles for NFAT3 and SMAD3 in OA pathogenesis and miR-140 transcription, potentially advancing OA therapeutic strategies.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) regulate gene expression, with miR-140 found in the WWP2 gene.
- miR-140 targets genes detrimental to osteoarthritis (OA), but its expression is reduced in OA chondrocytes.
- Investigated the regulation of miR-140 in human OA chondrocytes due to decreased expression.
Purpose of the Study:
- To elucidate the regulatory mechanisms of miR-140 in human OA chondrocytes.
- To identify factors influencing miR-140 expression independent of WWP2.
- To explore the roles of NFAT3 and SMAD3 in miR-140 regulation within the context of OA.
Main Methods:
- Quantitative polymerase chain reaction (qPCR) for gene expression analysis.
- Small interfering RNA (siRNA) for gene silencing in OA chondrocytes.
- Chromatin immunoprecipitation (ChIP) and mutagenesis to identify regulatory binding sites on rsmiR-140.
- Immunocytochemistry to assess protein translocation.
Main Results:
- WWP2 expression was similar in normal and OA cells, indicating miR-140 regulation independent of WWP2.
- NFAT3 and SMAD3 were identified as direct regulators of miR-140 transcription, acting as an activator and repressor, respectively.
- Transforming growth factor-beta (TGF-β) interfered with NFAT3 translocation, subsequently impacting miR-140 expression.
Conclusions:
- This study presents the first evidence of miR-140 regulation independent of WWP2.
- Identified distinct roles for NFAT3 and SMAD3 in regulating miR-140 transcription during OA.
- Findings offer potential for novel therapeutic strategies targeting OA by modulating miR-140 regulatory pathways.
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