MicroRNA-194 reciprocally stimulates osteogenesis and inhibits adipogenesis via regulating COUP-TFII expression

Abstract

Osteoblasts and adipocytes are differentiated from common mesenchymal stem cells (MSCs) in processes which are tightly controlled by various growth factors, signaling molecules, transcriptional factors and microRNAs. Recently, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) was identified as a critical regulator of MSC fate. In the present study, we aimed to identify some microRNAs (miR), which target COUP-TFII, and to determine the effects on MSCs fate. During osteoblastic or adipocytic differentiation from MSCs lineage cells, miR-194 expression was found to be reversal. In the cultures of mesenchymal C3H10T1/2 and primary bone marrow stromal cells, osteogenic stimuli increased miR-194 expression with accompanying decreases in COUP-TFII expression, whereas adipogenic stimuli reduced miR-194 expression with accompanying increases in COUP-TFII expression. A luciferase assay with COUP-TFII 3'-untranslated region (UTR) reporter plasmid, including the miR-194 binding sequences, showed that the introduction of miR-194 reduced the luciferase activity. However, it did not affect the activity of mutated COUP-TFII 3'-UTR reporter. Enforced expression of miR-194 significantly enhanced osteoblast differentiation, but inhibited adipocyte differentiation by decreasing COUP-TFII mRNA and protein levels. In contrast, inhibition of the endogenous miR-194 reduced matrix mineralization in the MSCs cultures, promoting the formation of lipid droplets by rescuing COUP-TFII expression. Furthermore, overexpression of COUP-TFII reversed the effects of miR-194 on the cell fates. Taken together, our results showed that miR-194 acts as a critical regulator of COUP-TFII, and can determinate the fate of MSCs to differentiate into osteoblasts and adipocytes. This suggests that miR-194 and COUP-TFII may be good target molecules for controlling bone and metabolic diseases.

Figure 1

Expression of miR-194 was induced during osteoblast differentiation and reduced during adipocyte differentiation. (a) Schematic representation of the eight miRNAs predicted to target COUP-TFII-3′-UTR. (b-d) Mesenchymal C3H10T1/2 cells were cultured in osteogenic induction medium (OM) or adipogenic induction medium (AM) for 3 days. (b) Expression levels of COUP-TFII and miRNAs as determined by qRT-PCR (n=3). After normalizing against β-actin or sno234, and compared with that of growth medium (GM) control. Values represent mean±S.D. *, P<0.05. (c) Relative expression was calculated after normalization to β-actin or sno234 levels (n=3). Values represent mean±S.D. *, P<0.05 and **, P<0.01. (d) Western blotting analysis was performed for protein levels of COUP-TFII. β-actin was used as a loading control. (e, f) Primary BMSCs were cultured in OM or AM for 4 days. The expression levels of miR-194 and COUP-TFII were analyzed by qRT-PCR. Relative expression was calculated after normalization to β-actin or sno234 levels (n=3). Values represent mean±S.D. *, P<0.05 compared with growth medium cultures. (g) Western blotting analysis was performed for protein levels of COUP-TFII. β-actin was used as a loading control

Figure 2

MiR-194 directly targets COUP-TFII mRNA. (a, b) C3H10T1/2 cells were transfected with miR-194 precursors or anti-miR-194. Expression levels of miR-194 and COUP-TFII mRNA were examined using qRT-PCR (n=3). Values represent mean±S.D. *, P<0.05 and **, P<0.01 compared with miR-CTL. (c) C3H10T1/2 cells were transfected with miR-194 precursors or anti-miR-194, and Western blotting analysis was performed. The data were obtained from three independent experiments. (d) Computational analysis of one putative complementary sequence for miR-194 in the 3′-UTR fragment of COUP-TFII. The wild type (WT) or mutant type (MT) construct was inserted into pmirGLO reporter vector. (e) The pmirGLO, pmirGLO COUP-TFII-3′-UTR-WT (WT) and pmirGLO COUP-TFII-3′-UTR-MT (MT) vectors were co-transfected with miR-CTL and miR-194 or anti-miR-194, respectively, into C3H10T1/2 cells. Luciferase activities were measured from the cell lysates after 24 h. Relative renilla luciferase activity was normalized to that of firefly luciferase. The values were normalized by the miR-CTL-treated group (n=3). **, P<0.01. All experiments were independently repeated at least three times

Figure 3

MiR-194 promotes osteoblasts differentiation. (a, b) Primary osteoblasts were cultured in OM for the indicated time period. Total RNA was isolated, and the expression levels of miR-194 and osteogenic markers were evaluated by qRT-PCR or RT-PCR, respectively (n=3). *, P<0.05 and **, P<0.01 compared with day 0 group. (c–f) C3H10T1/2 cells were transfected with miR-194 precursors or anti-miR-194 for 24 h, and then treated with OM for an additional 4-day period before performing RT-PCR (c, e) or Western blot analysis (d, f) with the indicated primers or antibodies. All data presented were independently repeated three times. (g, h) C3H10T1/2 cells were transfected with miR-194 precursor or anti-miR-194 for 24 h, and then treated with OM for an additional 7-day period before alizarin red staining was performed. A representative image of three independent experiments was shown

Figure 4

MiR-194 suppresses adipocytes differentiation. (a, b) 3T3-L1 cells were cultured in AM for the indicated time period, after which total RNA was isolated and expression levels of miR-194 and adipogenic markers were evaluated by qRT-PCR or RT-PCR, respectively (n=3). **, P < 0.01 compared with day 0 group. (c, e) 3T3-L1 cells were transfected with miR-194 precursors or anti-miR-194 for 24 h, and then cultured in AM for an additional 4-day period before performing RT-PCR with indicated primers, respectively (n=3). *, P<0.05 compared with the miR-CTL group. (d, f) Western blot analysis with indicated antibodies. (g, h) 3T3-L1 cells were transfected with miR-194 precursors or anti-miR-194 for 24 h and then treated with AM for an additional 7-day period before performing Oil Red O staining. A representative image of three independent experiments was shown

Figure 5

MiR-194 can regulate osteogenesis and adipogenesis from mesenchymal cells. (a) C3H10T1/2 cells were co-transfected with miR-194 (10 nM) or miR-CTL (10 nM) and COUP-TFII expression vector without 3′-UTR (100 ng) or control pcDNA3 vector (100 ng), cultured in OM for 6 days, and then subjected to alizarin red staining. The levels of calcium deposition was quantified by using image J software (n=3). *, P < 0.05 compared with the control group. ^#, P < 0.05 compared with the indicated group. (b) Expression levels of osteogenic markers were determined by RT-PCR after culturing in OM for 4 days. (c) 3T3-L1 cells were co-transfected with miR-194 (40 nM) or miR-con (40 nM) and the COUP-TFII expression vector (100 ng) or control pcDNA3 vector (100 ng), cultured in AM for 6 days and then subjected to Oil Red O staining. The levels of oil drop was quantified by using image J software (National Institutes of Health, Bethesda, MD, USA) (n=3). *, P < 0.05 and **, P < 0.01 compared with the control group. ^#, P < 0.05 compared with the indicated group. (d) Expression levels of adipogenic markers were determined by RT-PCR. (e) Schematic diagram of miR-194-mediated regulation of osteoblast and adipocyte differentiation from MSCs