The present study assessed the effects of microRNA-1 (miR-1) on the development of osteoarthritis using human tissues and a col2a1-cre-ER T2 /GFP fl/fl-RFP-miR-1 mouse model of osteoarthritis. Human cartilage tissues (n=20) were collected for reverse transcription-quantitative polymerase chain reaction (RT-qPcR), histological analysis and immunohistochemistry experiments. A transgenic mouse model of osteoarthritis was established by subjecting col2a1-cre-ER T2 /GFP fl/fl-RFP-miR-1 transgenic mice to anterior cruciate ligament transection (AcLT). Mice were subjected to radiography and in vivo fluorescence molecular tomography (FMT), while mouse tissues were collected for histological analysis, RT-qPcR and Safranin O staining. It was found that the miR-1 level was downregulated, whereas the levels of Indian hedgehog (Ihh), as well as those of its downstream genes were upregulated in human osteoarthritic cartilage. In the transgenic mice, treatment with tamoxifen induced miR-1, as well as collagen, type II (col2a1) and Aggrecan (Acan) expression; however, it decreased Ihh, glioma-associated oncogene homolog (Gli)1, Gli2, Gli3, smoothened homolog (Smo), matrix metalloproteinase (MMP)-13 and collagen type X (col10) expression. Safranin O staining revealed cartilage surface damage in the non-tamoxifen + AcLT group, compared with that in the tamoxifen + AcLT group. Histologically, an intact cartilage surface and less fibrosis were observed in the tamoxifen + AcLT group. Immunohistochemistry revealed that the protein expression of Ihh, col10, and MMP-13 was significantly higher in the joint tissues of the non-tamoxifen + AcLT group than in those of the tamoxifen + AcLT group. However, col2a1 expression was lower in the joint tissues of the non-tamoxifen + AcLT group than in those of the tamoxifen + AcLT group. The results of RT-qPcR and FMT further confirmed these findings. On the whole, the findings of the present study demonstrate that miR-1 expression protects against osteoarthritis-induced cartilage damage and gene expression by inhibiting Ihh signaling.
Background
The aim of this study was to evaluate whether histone deacetylase 4 S246/467/632A mutant (m-HDAC4) has enhanced function at histone deacetylase 4 (HDAC4) to attenuate cartilage degeneration in a rat model of osteoarthritis (OA).
Methods
Chondrocytes were infected with Ad-m-HDAC4-GFP or Ad-HDAC4-GFP for 24 h, incubated with interleukin-1β (IL-1β 10 ng/mL) for 24 h, and then measured by RT-qPCR. Male Sprague-Dawley rats (n = 48) were randomly divided into four groups and transduced with different vectors: ACLT/Ad-GFP, ACLT/Ad-HDAC4-GFP, ACLT/Ad-m-HDAC4-GFP, and sham/Ad-GFP. All rats received intra-articular injections 48 h after the operation and every 3 weeks thereafter. Cartilage damage was assessed using radiography and Safranin O staining and quantified using the OARSI score. The hypertrophic and anabolic molecules were detected by immunohistochemistry and RT-qPCR.
Results
M-HDAC4 decreased the expression levels of Runx-2, Mmp-13, and Col 10a1, but increased the levels of Col 2a1 and ACAN more effectively than HDAC4 in the IL-1β-induced chondrocyte OA model; upregulation of HDAC4 and m-HDAC4 in the rat OA model suppressed Runx-2 and MMP-13 production, and enhanced Col 2a1 and ACAN synthesis. Stronger Safranin O staining was detected in rats treated with m-HDAC4 than in those treated with HDAC4. The resulting OARSI scores were lower in the Ad-m-HDAC4 group (5.80 ± 0.45) than in the Ad-HDAC4 group (9.67 ± 1.83, P = 0.045). The OARSI scores were highest in rat knees that underwent ACLT treated with Ad-GFP control adenovirus vector (14.93 ± 2.14, P = 0.019 compared with Ad-HDAC4 group; P = 0.003 compared with Ad-m-HDAC4 group). Lower Runx-2 and MMP-13 production, and stronger Col 2a1 and ACAN synthesis were detected in rats treated with m-HDAC4 than in those treated with HDAC4.
Conclusions
M-HDAC4 repressed chondrocyte hypertrophy and induced chondrocyte anabolism in the nucleus. M-HDAC4 was more effective in attenuating articular cartilage damage than HDAC4.
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