MicroRNA-141 and -200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5 - PubMed (original) (raw)

MicroRNA-141 and -200a are involved in bone morphogenetic protein-2-induced mouse pre-osteoblast differentiation by targeting distal-less homeobox 5

Tomohiro Itoh et al. J Biol Chem. 2009.

Abstract

MicroRNAs (miRs) are endogenously expressed 18-25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it was indicated that miRs act as key regulators in cell differentiation, cell growth, and cell death. In osteogenesis, several miRs (for example miR-26a, -125b, -133, and -135) regulate osteoblast cell growth or differentiation in human adipose tissue-derived stem cells, mouse mesenchymal ST2 stem cells, and mouse premyogenic C2C12 cells. Additionally, Smad proteins control Drosha-mediated miR maturation. Therefore, miRs are closely related to osteogenesis. Here we investigated miR expression profile by an miR array and identified the candidate miRs, miR-141 and -200a, as pre-osteoblast differentiation-related miRs. The effects of miR-141 and -200a on pre-osteoblast differentiation were examined by using transfection of murine pre-osteoblastic MC3T3-E1 cells with mature miR-141 or -200a and antisense inhibitor for miR-141 or -200a. It was shown that miR-141 and -200a remarkably modulated the BMP-2-induced pre-osteoblast differentiation through the translational repression of Dlx5, which is a bone-generating transcription factor expressed in pre-osteoblast differentiation. Furthermore, it was indicated that Dlx5 is a common target of miR-141 and -200a by using a luciferase reporter assay. Thus, we have observed for the first time that miR-141 and -200a are involved in pre-osteoblast differentiation in part by regulating the expression of Dlx5.

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Figures

FIGURE 1.

FIGURE 1.

Down-regulation of miR-141 and -200a in BMP-2-induced pre-osteoblast differentiation. A, sequences of miR-141 and -200a and their complementation to Dlx5. B, changes of miR-141 and -200a expression levels in BMP-2-treated cells. MC3T3-E1 cells were treated with BMP-2 for 24, 48, and 72 h, and then total RNA was extracted from the cells by TRIzol with DNase I treatment. Expression levels of miR-141 and -200a in BMP-2-treated cells were examined by TaqMan® microRNA assay using a real time-PCR. The relative ratio is shown. The value of the cells without BMP-2 treatment was designated as 1.

FIGURE 2.

FIGURE 2.

Increased ALP activity after BMP-2 treatment in MC3T3-E1 cells. MC3T3-E1 cells were seeded into 24-well plated (1 × 105 cells/ml, 500 μl/well) and cultured with or without BMP-2 for 3 days. ALP activity was examined by a spectrophotometric method using a LabAssayTMALP kit. The values of each group were expressed as mean ± S.E. of three separate experiments. Values are significantly different as indicated. *, p < 0.01 by Student's t test. p < 0.05 is significant.

FIGURE 3.

FIGURE 3.

Effects of transfection with mature and antisense inhibitor for miR-141 or -200a on differentiation in MC3T3-E1 cells. A, differentiation and mineralization in MC3T3-E1 cells transfected with miR-141 or -200a were observed by ALP and Alizarin red staining. Negative control miR-NC (miR-NC) was designed to have no significant sequence similarity to mouse, rat, or human transcript sequences. B, increased expression of miR-141 or -200a by transfection significantly suppressed the ALP enzyme activity. Cells were treated with BMP-2 (300 ng/ml) for 3 days. The values of each group were expressed as mean ± S.E. of three separate experiments. Values are significantly different as indicated. *, p < 0.01 by Student's t test. p < 0.05 is significant. C, transfection of the cells with antisense inhibitor for miRNA-141 or -200a significantly increased the ALP activity. The values of each group were expressed as mean ± S.E. of three separate experiments. Significant differences are shown as follows: *, p < 0.1; **, p < 0.05; and ***, p < 0.01 by Student's t test. p < 0.05 is significant.

FIGURE 4.

FIGURE 4.

Expression of Dlx5 is translationally regulated by miR-141 and -200a in osteoblast differentiation. A, time course of Dlx5 and Osx expressions in BMP-2-treated MC3T3-E1 cells (20 μg of protein/lane). B, time course of Dlx5 and Osx mRNA expressions in BMP-2-treated MC3T3-E1 cells. C, transition of Dlx5 and Osx expressions in MC3T3-E1 cells transfected with miR-141 or -200a (treatment time: 72 h, 20 μg of protein/lane). D, changes of Dlx5 (panel i) and Osx (panel ii) mRNA expression in the transfected cells were examined by qRT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Panel iii, relative levels of Dlx5 mRNA in the transfected cells were normalized to glyceraldehyde-3-phosphate dehydrogenase. Lane 1, BMP-2 (−); lane 2, BMP-2 (+); lane 3, BMP-2(+) (300 ng/ml) and miR-negative control; lane 4, BMP-2(+) and miR-141(40 n

m

); lane 5, BMP-2(+) and miR-200a (40 n

m

). E, changes of Dlx5 and Osx protein expression in MC3T3-E1 cells transfected with antisense inhibitor miR-141 or -200a (treatment time: 72 h, 20 μg of protein/lane).

FIGURE 5.

FIGURE 5.

Identification of the target gene of miRNA-141/-200a by luciferase reporter assay. A, schematic representation of the sensor vectors used in the luciferase assay for identification of target region in Dlx5 for miR-141 and -200a. Construction of four kinds (A–D) of luciferase reporter plasmids is shown, including the 3′-UTR of Dlx5 mRNA. B, luciferase activities were measured by the using the luciferase assay system (Promega) according to the manufacturer's protocol. The luciferase activity per 1 μg of protein was measured, and the relative luciferase activities were expressed as the ratio of that in treated cells to that in the control cells. The activity value of miR-NC (nonspecific control) is expressed as 100% in each group. The values of each group were expressed as mean ± S.E. of three separate experiments. Values are significantly different as indicated. *, p < 0.01 by Student's t test. p < 0.05 is significant. C, mutation within or without the region corresponding to the seed sequence of miR-141 and -200a was made in each mutant from pGL3-Dlx5/miR-141 and -200a sensor-B. D, MC3T3-E1 cells were co-transfected with the pGL3-Dlx5/miR-141 and -200a sensor-B or mutated pGL3-Dlx5/miR-141 and -200a sensor-B (Mut1–3) and miR-141, -200a, or miR-NC. The activity of miR-NC (nonspecific control) is expressed as 100% in each group. The values of each group were expressed as mean ± S.E. of three separate experiments. Values are significantly different as indicated. *, p < 0.01 by Student's t test. p < 0.05 is significant.

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