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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.


Effects of siRNA-mediated silencing of HDAC4 on RUNX2, MMP13, and COL2A1 expression. (A–D) SW1353 cells were transfected with two different HDAC4-specific siRNA molecules for 6 h and then cultured for another 24 h. Subsequently, the mRNA expression levels of (A) HDAC4, (B) RUNX2, (C) MMP13, and (D) COL2A1 were measured by quantitative real-time reverse transcription (qRT)-PCR analysis; (E) SW1353 cells were transfected with the two HDAC4-specific siRNA molecules for 6 h and cultured for another 48 h. Cells were then harvested and subjected to Western blot analysis to evaluate the protein expression levels of HDAC4, RUNX2, MMP13 (active form), and β-actin; (F) graphic depiction of the protein expression levels quantified from the data presented in panel E. 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; MMP13, matrix metalloproteinase 13; COL2A1, type II collagen.
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ijms-17-01377-f004: Effects of siRNA-mediated silencing of HDAC4 on RUNX2, MMP13, and COL2A1 expression. (A–D) SW1353 cells were transfected with two different HDAC4-specific siRNA molecules for 6 h and then cultured for another 24 h. Subsequently, the mRNA expression levels of (A) HDAC4, (B) RUNX2, (C) MMP13, and (D) COL2A1 were measured by quantitative real-time reverse transcription (qRT)-PCR analysis; (E) SW1353 cells were transfected with the two HDAC4-specific siRNA molecules for 6 h and cultured for another 48 h. Cells were then harvested and subjected to Western blot analysis to evaluate the protein expression levels of HDAC4, RUNX2, MMP13 (active form), and β-actin; (F) graphic depiction of the protein expression levels quantified from the data presented in panel E. 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; MMP13, matrix metalloproteinase 13; COL2A1, type II collagen.

Mentions: To evaluate the effects of HDAC4 on RUNX2 and MMP13 expression, SW1353 cells were transfected with two HDAC4-specific siRNA molecules, siHDAC4-1 and siHDAC4-2, respectively. The depletion efficiencies for siHDAC4-1 and siHDAC4-2 were approximately 70% (Figure 4A), and knockdown of HDAC4 resulted in both increased RUNX2 and MMP13 mRNA expression and decreased COL2A1 mRNA expression (Figure 4B–D). Likewise, depletion of HDAC4 also yielded increased RUNX2 and active MMP13 protein expression (Figure 4E,F). Therefore, the effect of HDAC4 siRNA was, to some extent, similar to that of miR-381. These findings further support the hypothesis that miR-381 represses HDAC4.


MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression
Effects of siRNA-mediated silencing of HDAC4 on RUNX2, MMP13, and COL2A1 expression. (A–D) SW1353 cells were transfected with two different HDAC4-specific siRNA molecules for 6 h and then cultured for another 24 h. Subsequently, the mRNA expression levels of (A) HDAC4, (B) RUNX2, (C) MMP13, and (D) COL2A1 were measured by quantitative real-time reverse transcription (qRT)-PCR analysis; (E) SW1353 cells were transfected with the two HDAC4-specific siRNA molecules for 6 h and cultured for another 48 h. Cells were then harvested and subjected to Western blot analysis to evaluate the protein expression levels of HDAC4, RUNX2, MMP13 (active form), and β-actin; (F) graphic depiction of the protein expression levels quantified from the data presented in panel E. 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; MMP13, matrix metalloproteinase 13; COL2A1, type II collagen.
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ijms-17-01377-f004: Effects of siRNA-mediated silencing of HDAC4 on RUNX2, MMP13, and COL2A1 expression. (A–D) SW1353 cells were transfected with two different HDAC4-specific siRNA molecules for 6 h and then cultured for another 24 h. Subsequently, the mRNA expression levels of (A) HDAC4, (B) RUNX2, (C) MMP13, and (D) COL2A1 were measured by quantitative real-time reverse transcription (qRT)-PCR analysis; (E) SW1353 cells were transfected with the two HDAC4-specific siRNA molecules for 6 h and cultured for another 48 h. Cells were then harvested and subjected to Western blot analysis to evaluate the protein expression levels of HDAC4, RUNX2, MMP13 (active form), and β-actin; (F) graphic depiction of the protein expression levels quantified from the data presented in panel E. 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; MMP13, matrix metalloproteinase 13; COL2A1, type II collagen.
Mentions: To evaluate the effects of HDAC4 on RUNX2 and MMP13 expression, SW1353 cells were transfected with two HDAC4-specific siRNA molecules, siHDAC4-1 and siHDAC4-2, respectively. The depletion efficiencies for siHDAC4-1 and siHDAC4-2 were approximately 70% (Figure 4A), and knockdown of HDAC4 resulted in both increased RUNX2 and MMP13 mRNA expression and decreased COL2A1 mRNA expression (Figure 4B–D). Likewise, depletion of HDAC4 also yielded increased RUNX2 and active MMP13 protein expression (Figure 4E,F). Therefore, the effect of HDAC4 siRNA was, to some extent, similar to that of miR-381. These findings further support the hypothesis that miR-381 represses HDAC4.

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.