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MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9.

Yang B, Guo H, Zhang Y, Chen L, Ying D, Dong S - PLoS ONE (2011)

Bottom Line: In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels.Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes.Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing, People's Republic of China.

ABSTRACT
Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3'-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.

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Mir-145 represses the expression of Sox9 at protein level in early stage of chondrogenic differentiation.(A) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control individually. After induced by TGF-β3 for 24 h, 7 d and 14 d, the cells were harvested for measurement of Sox9 protein expression using Western blot. β-actin acts as an internal control. Quantitation of the Sox9 protein level was performed using Quantity One software. The result is shown in the below panels. (B) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control, and then mRNA level of Sox9 were measured using qRT-PCR at 24h. β-actin acts as an internal control in qRT-PCR analysis. The relative expression level of Sox9 mRNA in cells transfected with control oligonucleotide was set to one, as control. Three independent experiments were done and data was represented as mean±sd.
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pone-0021679-g003: Mir-145 represses the expression of Sox9 at protein level in early stage of chondrogenic differentiation.(A) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control individually. After induced by TGF-β3 for 24 h, 7 d and 14 d, the cells were harvested for measurement of Sox9 protein expression using Western blot. β-actin acts as an internal control. Quantitation of the Sox9 protein level was performed using Quantity One software. The result is shown in the below panels. (B) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control, and then mRNA level of Sox9 were measured using qRT-PCR at 24h. β-actin acts as an internal control in qRT-PCR analysis. The relative expression level of Sox9 mRNA in cells transfected with control oligonucleotide was set to one, as control. Three independent experiments were done and data was represented as mean±sd.

Mentions: The different degree of complementary between miRNA and its target mRNA probably determines that miRNA repress target mRNA through two distinct pathways. MiRNA suppresses mRNA translation bearing imperfect complementary target sequences and degrades mRNA bearing perfect complementary target sequences[41], [42], [43]. Computational algorithms predicted that miR-145 would bind to the Sox9 3′-UTR with imperfect complementation, suggesting that it may not result in Sox9 mRNA cleavage. To demonstrate whether miR-145 acts as attenuator of Sox9 protein expression, we transfected C3H10T1/2 mesenchymal stem cells with either pre-miR-145 or anti-miR-145 for 24 h, and then exposed the transfected cells to the chondrogenic differentiation medium primarily consisting of TGF-β3. We firstly performed the non-transfected control in preliminary experiment for optimizing the condition of transfection with miRNAs. At the optimal condition of transfection, there is no significant difference of Sox9 protein expression between tansfected group and non-transfected control (Figure S1). As expected for the mechanisms of miRNAs regulation, measured by western blot assay, Sox9 protein level was notably decreased in the cells of miR-145 over-expression and increased in the cells of miR-145 suppression at 1 d and 7 d but not at 14 d by TGF-β3 treatment (Figure 3A). However, qRT-PCR analysis showed no significant change in Sox9 mRNA level in both transfected cells (Figure 3B-). Thus, these data demonstrate miR-145 inhibits Sox9 protein expression but not mRNA levels in mesenchymal stem cell line at early stage of chondrogenic differentiation.


MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9.

Yang B, Guo H, Zhang Y, Chen L, Ying D, Dong S - PLoS ONE (2011)

Mir-145 represses the expression of Sox9 at protein level in early stage of chondrogenic differentiation.(A) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control individually. After induced by TGF-β3 for 24 h, 7 d and 14 d, the cells were harvested for measurement of Sox9 protein expression using Western blot. β-actin acts as an internal control. Quantitation of the Sox9 protein level was performed using Quantity One software. The result is shown in the below panels. (B) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control, and then mRNA level of Sox9 were measured using qRT-PCR at 24h. β-actin acts as an internal control in qRT-PCR analysis. The relative expression level of Sox9 mRNA in cells transfected with control oligonucleotide was set to one, as control. Three independent experiments were done and data was represented as mean±sd.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140487&req=5

pone-0021679-g003: Mir-145 represses the expression of Sox9 at protein level in early stage of chondrogenic differentiation.(A) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control individually. After induced by TGF-β3 for 24 h, 7 d and 14 d, the cells were harvested for measurement of Sox9 protein expression using Western blot. β-actin acts as an internal control. Quantitation of the Sox9 protein level was performed using Quantity One software. The result is shown in the below panels. (B) C3H10T1/2 cells were transfected with pre-miR-145, anti-miR-145 or their control, and then mRNA level of Sox9 were measured using qRT-PCR at 24h. β-actin acts as an internal control in qRT-PCR analysis. The relative expression level of Sox9 mRNA in cells transfected with control oligonucleotide was set to one, as control. Three independent experiments were done and data was represented as mean±sd.
Mentions: The different degree of complementary between miRNA and its target mRNA probably determines that miRNA repress target mRNA through two distinct pathways. MiRNA suppresses mRNA translation bearing imperfect complementary target sequences and degrades mRNA bearing perfect complementary target sequences[41], [42], [43]. Computational algorithms predicted that miR-145 would bind to the Sox9 3′-UTR with imperfect complementation, suggesting that it may not result in Sox9 mRNA cleavage. To demonstrate whether miR-145 acts as attenuator of Sox9 protein expression, we transfected C3H10T1/2 mesenchymal stem cells with either pre-miR-145 or anti-miR-145 for 24 h, and then exposed the transfected cells to the chondrogenic differentiation medium primarily consisting of TGF-β3. We firstly performed the non-transfected control in preliminary experiment for optimizing the condition of transfection with miRNAs. At the optimal condition of transfection, there is no significant difference of Sox9 protein expression between tansfected group and non-transfected control (Figure S1). As expected for the mechanisms of miRNAs regulation, measured by western blot assay, Sox9 protein level was notably decreased in the cells of miR-145 over-expression and increased in the cells of miR-145 suppression at 1 d and 7 d but not at 14 d by TGF-β3 treatment (Figure 3A). However, qRT-PCR analysis showed no significant change in Sox9 mRNA level in both transfected cells (Figure 3B-). Thus, these data demonstrate miR-145 inhibits Sox9 protein expression but not mRNA levels in mesenchymal stem cell line at early stage of chondrogenic differentiation.

Bottom Line: In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels.Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes.Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing, People's Republic of China.

ABSTRACT
Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3'-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.

Show MeSH
Related in: MedlinePlus