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MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression

View Article: PubMed Central - PubMed

ABSTRACT

Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3′-untranslated region (3′-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.

No MeSH data available.


Evaluation of the mRNA and protein expression levels of HDAC4 and RUNX2 after overexpression/knockdown of miR-381. (A,B) SW1353 cells were transfected with (A) a miR-381 mimic or (B) a miR-381 inhibitor and the mRNA expression levels of HDAC4 and RUNX2 were determined by quantitative real-time reverse transcription (qRT)-PCR analysis; (C–F) Western blot analysis of HDAC4 and RUNX2 expression in SW1353 cells transfected with (C,D) a miR-381 mimic or (E,F) a miR-381 inhibitor. Panels D and F contain graphic depictions of the protein expression levels quantified from the data presented in panels C and E, respectively. Data are presented as means ± standard deviations from three independent experiments. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2.
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ijms-17-01377-f003: Evaluation of the mRNA and protein expression levels of HDAC4 and RUNX2 after overexpression/knockdown of miR-381. (A,B) SW1353 cells were transfected with (A) a miR-381 mimic or (B) a miR-381 inhibitor and the mRNA expression levels of HDAC4 and RUNX2 were determined by quantitative real-time reverse transcription (qRT)-PCR analysis; (C–F) Western blot analysis of HDAC4 and RUNX2 expression in SW1353 cells transfected with (C,D) a miR-381 mimic or (E,F) a miR-381 inhibitor. Panels D and F contain graphic depictions of the protein expression levels quantified from the data presented in panels C and E, respectively. Data are presented as means ± standard deviations from three independent experiments. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2.

Mentions: The divergent expression of miR-381 and HDAC4 observed during late-stage chondrogenesis of ATDC5 cells and in the long bones of mouse embryos implies that miR-381 might directly inhibit HDAC4 expression. To address this hypothesis, HDAC4 mRNA and protein expression levels were evaluated in SW1353 cells transfected with a miR-381 mimic, or a miR-381 inhibitor by quantitative real-time reverse transcription (qRT-PCR) and Western blot analyses, respectively. While cells overexpressing miR-381 exhibited significantly decreased mRNA and protein expression of HDAC4, those treated with the miR-381 inhibitor showed increased HDAC4 expression (Figure 3). Conversely, overexpression and inhibition of miR-381 resulted in markedly increased and decreased RUNX2 mRNA and protein expression, respectively (Figure 3).


MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression
Evaluation of the mRNA and protein expression levels of HDAC4 and RUNX2 after overexpression/knockdown of miR-381. (A,B) SW1353 cells were transfected with (A) a miR-381 mimic or (B) a miR-381 inhibitor and the mRNA expression levels of HDAC4 and RUNX2 were determined by quantitative real-time reverse transcription (qRT)-PCR analysis; (C–F) Western blot analysis of HDAC4 and RUNX2 expression in SW1353 cells transfected with (C,D) a miR-381 mimic or (E,F) a miR-381 inhibitor. Panels D and F contain graphic depictions of the protein expression levels quantified from the data presented in panels C and E, respectively. Data are presented as means ± standard deviations from three independent experiments. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2.
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ijms-17-01377-f003: Evaluation of the mRNA and protein expression levels of HDAC4 and RUNX2 after overexpression/knockdown of miR-381. (A,B) SW1353 cells were transfected with (A) a miR-381 mimic or (B) a miR-381 inhibitor and the mRNA expression levels of HDAC4 and RUNX2 were determined by quantitative real-time reverse transcription (qRT)-PCR analysis; (C–F) Western blot analysis of HDAC4 and RUNX2 expression in SW1353 cells transfected with (C,D) a miR-381 mimic or (E,F) a miR-381 inhibitor. Panels D and F contain graphic depictions of the protein expression levels quantified from the data presented in panels C and E, respectively. Data are presented as means ± standard deviations from three independent experiments. * p < 0.05; ** p < 0.001. miR-381, microRNA-381; HDAC4, histone deacetylase 4; RUNX2, Runt-related transcription factor 2.
Mentions: The divergent expression of miR-381 and HDAC4 observed during late-stage chondrogenesis of ATDC5 cells and in the long bones of mouse embryos implies that miR-381 might directly inhibit HDAC4 expression. To address this hypothesis, HDAC4 mRNA and protein expression levels were evaluated in SW1353 cells transfected with a miR-381 mimic, or a miR-381 inhibitor by quantitative real-time reverse transcription (qRT-PCR) and Western blot analyses, respectively. While cells overexpressing miR-381 exhibited significantly decreased mRNA and protein expression of HDAC4, those treated with the miR-381 inhibitor showed increased HDAC4 expression (Figure 3). Conversely, overexpression and inhibition of miR-381 resulted in markedly increased and decreased RUNX2 mRNA and protein expression, respectively (Figure 3).

View Article: PubMed Central - PubMed

ABSTRACT

Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3&prime;-untranslated region (3&prime;-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.

No MeSH data available.