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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.


miR-381 modulates HDAC4 expression via interaction with a binding site within the 3′ untranslated region (UTR) of the HDAC4 mRNA; (A) sequences of the putative miR-381 binding sites within the 3′-UTR of HDAC4; (B,C) SW1353 cells were transfected with the wild-type HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR) alone (blank), or were co-transfected with Luc-HDAC4-3′-UTR or the mutated HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR-mut) and the miR-381 mimic or nonspecific control microRNA (miR-control). Luciferase activity was then assessed using the Dual-Glo Luciferase Assay System. Data are presented as means ± standard deviations from three independent experiments. ** p < 0.001. NS, not significant.
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ijms-17-01377-f005: miR-381 modulates HDAC4 expression via interaction with a binding site within the 3′ untranslated region (UTR) of the HDAC4 mRNA; (A) sequences of the putative miR-381 binding sites within the 3′-UTR of HDAC4; (B,C) SW1353 cells were transfected with the wild-type HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR) alone (blank), or were co-transfected with Luc-HDAC4-3′-UTR or the mutated HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR-mut) and the miR-381 mimic or nonspecific control microRNA (miR-control). Luciferase activity was then assessed using the Dual-Glo Luciferase Assay System. Data are presented as means ± standard deviations from three independent experiments. ** p < 0.001. NS, not significant.

Mentions: To clarify the molecular mechanism by which miR-381 influences HDAC4 expression, we analyzed the 3′-UTR sequence of the human HDAC4 mRNA using the Targetscan algorithm (Rlease 6.2, Whitehead Institute, Cambridge, MA, USA) and identified a potential miR-381 binding site (UUGUAU) (Figure 5A). Subsequently, to determine whether miR-381 directly modulates HDAC4 expression via interaction with this binding site, we performed luciferase reporter assays using SW1353 cells transfected with vectors harboring the wild-type or mutated 3′-UTR of HDAC4, in the presence or absence of the miR-381 or negative control (NC) mimic. The mutated 3′-UTR sequence was constructed to prevent the binding of the miR-381 mimic (Figure 5A). Compared to cells harboring only the HDAC4 3′-UTR luciferase reporter and cells co-transfected with the reporter vector and the NC mimic, those co-transfected with the reporter vector and the miR-381 mimic exhibited significantly reduced luciferase activity (Figure 5B). In contrast, no significant change in luciferase activity was noted in cells co-transfected with the miR-381 mimic and the luciferase reporter vector containing the mutated 3′-UTR (Figure 5C). These results indicate that miR-381 modulates the expression of HDAC4 by binding to the 3′-UTR of HDAC4 mRNA.


MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression
miR-381 modulates HDAC4 expression via interaction with a binding site within the 3′ untranslated region (UTR) of the HDAC4 mRNA; (A) sequences of the putative miR-381 binding sites within the 3′-UTR of HDAC4; (B,C) SW1353 cells were transfected with the wild-type HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR) alone (blank), or were co-transfected with Luc-HDAC4-3′-UTR or the mutated HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR-mut) and the miR-381 mimic or nonspecific control microRNA (miR-control). Luciferase activity was then assessed using the Dual-Glo Luciferase Assay System. Data are presented as means ± standard deviations from three independent experiments. ** p < 0.001. NS, not significant.
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ijms-17-01377-f005: miR-381 modulates HDAC4 expression via interaction with a binding site within the 3′ untranslated region (UTR) of the HDAC4 mRNA; (A) sequences of the putative miR-381 binding sites within the 3′-UTR of HDAC4; (B,C) SW1353 cells were transfected with the wild-type HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR) alone (blank), or were co-transfected with Luc-HDAC4-3′-UTR or the mutated HDAC4 3′-UTR luciferase reporter plasmid (Luc-HDAC4-3′-UTR-mut) and the miR-381 mimic or nonspecific control microRNA (miR-control). Luciferase activity was then assessed using the Dual-Glo Luciferase Assay System. Data are presented as means ± standard deviations from three independent experiments. ** p < 0.001. NS, not significant.
Mentions: To clarify the molecular mechanism by which miR-381 influences HDAC4 expression, we analyzed the 3′-UTR sequence of the human HDAC4 mRNA using the Targetscan algorithm (Rlease 6.2, Whitehead Institute, Cambridge, MA, USA) and identified a potential miR-381 binding site (UUGUAU) (Figure 5A). Subsequently, to determine whether miR-381 directly modulates HDAC4 expression via interaction with this binding site, we performed luciferase reporter assays using SW1353 cells transfected with vectors harboring the wild-type or mutated 3′-UTR of HDAC4, in the presence or absence of the miR-381 or negative control (NC) mimic. The mutated 3′-UTR sequence was constructed to prevent the binding of the miR-381 mimic (Figure 5A). Compared to cells harboring only the HDAC4 3′-UTR luciferase reporter and cells co-transfected with the reporter vector and the NC mimic, those co-transfected with the reporter vector and the miR-381 mimic exhibited significantly reduced luciferase activity (Figure 5B). In contrast, no significant change in luciferase activity was noted in cells co-transfected with the miR-381 mimic and the luciferase reporter vector containing the mutated 3′-UTR (Figure 5C). These results indicate that miR-381 modulates the expression of HDAC4 by binding to the 3′-UTR of HDAC4 mRNA.

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.