MicroRNA-1 regulates chondrocyte phenotype by repressing histone deacetylase 4 during growth plate development

Abstract

MicroRNAs (miRs) are noncoding RNAs (17-25 nt) that control translation and/or mRNA degradation. Using Northern blot analysis, we identified that miR-1 is specifically expressed in growth plate cartilage in addition to muscle tissue, but not in brain, intestine, liver, or lung. We obtained the first evidence that miR-1 is highly expressed in the hypertrophic zone of the growth plate, with an 8-fold increase compared with the proliferation zone; this location coincides with the Ihh and Col X expression regions in vivo. MiR-1 significantly induces chondrocyte proliferation and differentiation. We further identified histone deacetylase 4 (HDAC4) as a target of miR-1. HDAC4 negatively regulates chondrocyte hypertrophy by inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. MiR-1 inhibits both endogenous HDAC4 protein by 2.2-fold and the activity of a reporter gene bearing the 3'-untranslated region (UTR) of HDAC4 by 3.3-fold. Conversely, knockdown of endogenous miR-1 relieves the repression of HDAC4. Deletion of the miR-1 binding site in HDAC4 3'-UTR or mutated miR-1 abolishes miR-1-mediated inhibition of the reporter gene activity. Overexpression of HDAC4 reverses miR-1 induction of chondrocyte differentiation markers Col X and Ihh. HDAC4 inhibits Runx2 promoter activity in a dosage-dependent manner. Thus, miR-1 plays an important role in the regulation of the chondrocyte phenotype during the growth plate development via direct targeting of HDAC4.

Keywords: HDAC4; Runx2; collagen X; differentiation.

Figure 6.

Repression of HDAC4 3′-UTR by miR-1. A) Sequences of the miR-1 target sites in the 3′-UTR of HDAC4 in different species. MiR-1 is conserved in different mammalian species. The predicted seeding sites were shown in HDAC4 3′-UTR (position: 2334–2340, 3514–3520, 3547–3553). B) COS-1 cell was cotransfected with different deleted binding sites of HDAC4 3′-UTR plasmid and/or miR-1 mimic: 1) wild type containing 3 binding sites, 2) wild type+miR-1 (120 nM), 3) binding sites 1 and 2 deleted + miR-1 (120 nM), 4) binding sites 1 and 3 deleted + miR-1 (120 nM), 5) binding sites 2 and 3 deleted + miR-1 (120 nM), and 6) all three binding sites deleted + miR-1 (120 nM). Forty-eight hours after transfection, the cells were harvested for quantification of dual-luciferase activities. Data are the means of 3 independent experiments, with sd indicated. *P < 0.05, compared with HDAC4 wild type. C) COS-1 cell was cotransfected with pGL3-HDAC4 3′-UTR luciferase report and miR-1 or miR-31 or miR-1mut. Luciferase activity was determined 48 h after transfection. Data are the means of 3 independent experiments, with sd indicated. *P < 0.05.

Figure 1.

Expression of miR-1 in different tissues. A) Northern blot detected miR-1 in mouse heart, human articular cartilage, and chicken growth plate from d 17 embryos, but not in mouse brain, lung, intestine, or liver. B) The stained gel of total RNA showed 28S/18S ladder as loading control. C) RT-PCR detected miR-1 in mouse heart, human articular cartilage, and chicken growth plate from d 17 embryos, but not in other tissues. D) U6 was used as a loading control.

Figure 2.

MiR-1 is highly expressed in hypertrophic chondrocytes. A) Proliferative and hypertrophic zones were isolated from d 17 chicken tibia growth plates, and the middle part was discarded. B, C) Total RNA was isolated from the proliferative (pro) and hypertrophic (hyper) zones of tibia growth plate from d 17 chicken embryos. The expression levels of miR-1 were qualified by RT-PCR and Northern blot. U6 and RNU48 were used as internal control. D) The expression levels of miR-1 were quantified by real-time RT-PCR in the mouse proliferative and hypertrophic chondrocyte cell line (MCT cells). miR-133 in hypertrophic cells maintained a similar level of expression compared to proliferation chondrocytes. E) Real-time RT-PCR confirmed a higher expression of Col X and Ihh in the hypertrophic zone compared with the proliferative zone. Values represent the mean ± sd from 3 independent experiments. *P < 0.05. F) RT-PCR confirmed a high expression of Col 2 but not ACTA1 and MYOD1 (markers for skeletal muscle) was detected from the growth plate preparations. G) A high expression of miR-1 was detected in the prehypertrophic and hypertrophic zones compared with the proliferation zone by ISH.

Figure 3.

MiR-1 stimulates chondrocyte proliferation. A) More than 88% of chondrocytes were transfected with miR-1, as determined by immunofluorescence microscopy 24 h after transfection in the primary chicken chondrocytes. B) Real-time RT-PCR result showed that overexpression of miR-1 (120 nM) increases the level of miR-1 while knockdown miR-1 by anti-miR-1 decreases its expression compared to control miRNA-1 in the primary chicken chondrocytes 24 h post-transfection. Values are presented as mean ± sd (n=3). *P < 0.05. C, D) MiR-1 stimulates chondrocyte proliferation. Primary chicken chondrocytes were transfected with a miR-1 mimic (miR-1) or negative control mimic (ConmiR), and miR-1 inhibitor (anti-miR-1) or a control miRNA inhibitor (control) at 120 nM, respectively. Forty-eight hours post-transfection, cell growth was measured by an EDU-based cell proliferation assay. Data are presented as means ± sd (n=5). *P < 0.05. E) Proliferation of chicken chondrocytes was also measured by a CCK-8-based cell proliferation assay. Values are means ± sd (n=5). *P < 0.05. F) miR-1 mimic increases cell proliferation, while cells were cotransfected with miR-1 mimic and HDAC4 has an opposite effect. Data are presented as means ± sd (n=3). *P < 0.05, compared to ConmiR. ^#P < 0.05, compared to miR-1.

Figure 4.

MiR-1 induces chondrocyte differentiation. A) miR-1 increases the mRNA levels of Col X and Ihh, markers for chondrocyte differentiation. Primary chicken chondrocytes were transfected with a miR-1 mimic (miR-1) or control miRNA (ConmiR) at 120 nmol, respectively. The mRNA levels of Col X and Ihh were quantified by real-time RT-PCR 24 h post-transfection. Data are presented as mean ± sd (n=3) *P < 0.05. B) Inhibition of miR-1 down-regulates mRNA levels of Col X and Ihh. Primary chicken chondrocytes were transfected with a miR-1 inhibitor (anti-miR-1) or a control microRNA inhibitor (Control) at 120 nmol, respectively. The mRNA levels of Ihh and Col X were quantified by real-time RT-PCR 24 h post-transfection. Data are presented as means ± sd (n=3; 3 independent cell culture experiments). *P < 0.05. All transfections were performed in triplicate.

Figure 5.

MiR-1 down-regulates HDAC4 protein in chondrocytes. A) MiR-1 down-regulates HDAC4 protein in dosage-dependent manner. Chondrocytes from d 17 embryos of chicken growth plate were transfected with a miR-1 mimic (miR-1) at 80 and 120 nmol or with a control miRNA (ConmiR) at 120 nmol. Western blot analysis was performed with HDAC4 antibody 48 h after transfection. B) Chicken growth plate chondrocytes from d 17 embryos were transfected with a miR-1 mimic (miR-1) or control microRNA (ConmiR), and a miR-1 inhibitor (anti-miR-1) or a control microRNA inhibitor (Control) at 120 nmol, respectively. Western blot analysis was performed with an antibody against HDAC4 48 h after transfection. miR-1 (120 nM) reduces the level of HDAC4, while antimiR-1 increases the level of HDAC4 compared to the control. C) Band intensities obtained from 3 individual Western blots were quantified using ImageJ software after normalize to β-actin to generate a bar graph of the relative difference in HDAC4 protein levels. Error bars represent ±1 sd of the mean. *P < 0.05.

Figure 7.

MiR-1 increases Runx2 gene expression and Runx2 luciferase reporter activity. A) Chicken growth plate chondrocytes from d 17 embryos were transfected with a miR-1 mimic (miR-1) or a control miRNA (ConmiR), and a miR-1 inhibitor (anti-miR-1) or a control miRNA inhibitor (Control) at 120 nmol. Total RNA was extracted, and the mRNA level of Runx2 was quantified by real-time RT-PCR 24 h post-transfection. Data are the means of 3 independent experiments, with sd indicated. *P < 0.05, compared with control miR. B) Chondrocytes were transiently transfected with Runx2 promoter and expression vectors encoding HDAC4, as indicated in the presence or absence of control miRNA (ConmiR) or miR-1 mimic (miR-1). The cells were harvested for quantification of dual-luciferase activities 48 h after transfection. miR-1 increases Runx2 promoter activity while HDAC4 overcomes the Runx2 promoter activity induced by miR-1. Data are the means of 3 independent experiments, with sd indicated. *P < 0.05, compared with Con-miR; ^#P < 0.05, compared with miR-1 alone. C) HDAC4 inhibits Runx2 promoter activity in a dose-dependent manner. Data are the means of 3 independent cell culture experiments, with sd indicated. ^#P < 0.05, compared to control; *P < 0.05, compared to Runx 2 promoter alone. D) Knockdown HDAC4 by HDAC4 siRNA increases Runx2 promoter activity, while anti-miR-1 overcomes the Runx2 promoter activity induced by HDAC4 siRNA. Data are the means of 3 independent cell culture experiments, with sd indicated. *P < 0.05, compared to ConmiR; ^#P < 0.05, compared to HDAC4 siRNA alone.

Figure 8.

HDAC4 inhibiting miR-1 induction of Ihh and Col X expression. A) Chicken growth plate chondrocytes from d 17 embryo were transfected with a control miRNA (ConmiR) or miR-1 or miR-1 plus HDAC4 expression vector. The mRNA levels of Ihh and Col X were quantified by real-time RT-PCR 24 h after transfection. Data are the means of 3 independent experiments, with sd indicated. *P < 0.05, compared with ConmiR. #P < 0.05, compared with miR-1. B) Overexpression of HDAC4 increased HDAC4 protein levels. Western blot analysis was performed with cell lysates collected 48 h post-transfection by an antibody against HDAC4.