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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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Suppression of mir-145 enhances early chondrogenic differentiation of C3H10T1/2 cells.C3H10T1/2 cells were transfected with anti-miR-145 or its control respectively. (A) After 24 h, 7 d and 14 d of treatment with TGF-β3, all of cells were lysed and then the expression of chondrogenic differentiation markers, such as Col2a1, COMP, Agc1, Col9a2 and Col11a1, were measured via qRT-PCR. The relative expression level of mRNA in cells transfected with control oligonucleotide was set to one, as control. (B) After 3 d, 7 d and 14d of treatment with TGF-β3, all of pellets were measured by alcian blue staining. Three independent cell culture experiments were done and data was represented as mean±sd. *, p<0.05, when compared with control.
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pone-0021679-g005: Suppression of mir-145 enhances early chondrogenic differentiation of C3H10T1/2 cells.C3H10T1/2 cells were transfected with anti-miR-145 or its control respectively. (A) After 24 h, 7 d and 14 d of treatment with TGF-β3, all of cells were lysed and then the expression of chondrogenic differentiation markers, such as Col2a1, COMP, Agc1, Col9a2 and Col11a1, were measured via qRT-PCR. The relative expression level of mRNA in cells transfected with control oligonucleotide was set to one, as control. (B) After 3 d, 7 d and 14d of treatment with TGF-β3, all of pellets were measured by alcian blue staining. Three independent cell culture experiments were done and data was represented as mean±sd. *, p<0.05, when compared with control.

Mentions: To explore whether miR-145 has an effect on chondrogenic differentiation, we transfected either pre-miR-145 or anti-miR-145 into C3H10T1/2 cells. After induction of chondrogenic differentiation by medium containing TGF-β3 for 1 d and 7 d, qRT-PCR analysis of C3H10T1/2 cells transfected with pre-miR-145 showed a significant decrease in the mRNA expression levels of chondrogenesis markers including Col2a1, Agc1, COMP , Col9a2 and Col11a1 (Figure 4A). Moreover, alcian blue staining intensity were decreased following pre-miR-145 treatment for 3 d and 7 d (Figure 4B). These results reveal that miR-145 over-expression inhibits early chondrogenic differentiation. Inhibition of endogenous miR-145 expression in C3H10T1/2 cells by transfection of anti-miR-145, under the same induction conditions as above, resulted in enhancing chondrogenic differentiation as shown by a significant increase in chondrogenesis markers at mRNA level (Figure 5A) and alcian blue staining intensity (Figure 5B). The results of qRT-PCR and alcian blue staining have shown that modulation of miR-145 effected the expression of genes and GAGs related to chondrocyte after induced for 7 d. However, the same effect did not last for 14 d (Figure 4, 5). Collectively, our data demonstrate that miR-145 act as a key negative regulator of early 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)

Suppression of mir-145 enhances early chondrogenic differentiation of C3H10T1/2 cells.C3H10T1/2 cells were transfected with anti-miR-145 or its control respectively. (A) After 24 h, 7 d and 14 d of treatment with TGF-β3, all of cells were lysed and then the expression of chondrogenic differentiation markers, such as Col2a1, COMP, Agc1, Col9a2 and Col11a1, were measured via qRT-PCR. The relative expression level of mRNA in cells transfected with control oligonucleotide was set to one, as control. (B) After 3 d, 7 d and 14d of treatment with TGF-β3, all of pellets were measured by alcian blue staining. Three independent cell culture experiments were done and data was represented as mean±sd. *, p<0.05, when compared with control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140487&req=5

pone-0021679-g005: Suppression of mir-145 enhances early chondrogenic differentiation of C3H10T1/2 cells.C3H10T1/2 cells were transfected with anti-miR-145 or its control respectively. (A) After 24 h, 7 d and 14 d of treatment with TGF-β3, all of cells were lysed and then the expression of chondrogenic differentiation markers, such as Col2a1, COMP, Agc1, Col9a2 and Col11a1, were measured via qRT-PCR. The relative expression level of mRNA in cells transfected with control oligonucleotide was set to one, as control. (B) After 3 d, 7 d and 14d of treatment with TGF-β3, all of pellets were measured by alcian blue staining. Three independent cell culture experiments were done and data was represented as mean±sd. *, p<0.05, when compared with control.
Mentions: To explore whether miR-145 has an effect on chondrogenic differentiation, we transfected either pre-miR-145 or anti-miR-145 into C3H10T1/2 cells. After induction of chondrogenic differentiation by medium containing TGF-β3 for 1 d and 7 d, qRT-PCR analysis of C3H10T1/2 cells transfected with pre-miR-145 showed a significant decrease in the mRNA expression levels of chondrogenesis markers including Col2a1, Agc1, COMP , Col9a2 and Col11a1 (Figure 4A). Moreover, alcian blue staining intensity were decreased following pre-miR-145 treatment for 3 d and 7 d (Figure 4B). These results reveal that miR-145 over-expression inhibits early chondrogenic differentiation. Inhibition of endogenous miR-145 expression in C3H10T1/2 cells by transfection of anti-miR-145, under the same induction conditions as above, resulted in enhancing chondrogenic differentiation as shown by a significant increase in chondrogenesis markers at mRNA level (Figure 5A) and alcian blue staining intensity (Figure 5B). The results of qRT-PCR and alcian blue staining have shown that modulation of miR-145 effected the expression of genes and GAGs related to chondrocyte after induced for 7 d. However, the same effect did not last for 14 d (Figure 4, 5). Collectively, our data demonstrate that miR-145 act as a key negative regulator of early 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.

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Related in: MedlinePlus