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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 directly targets Sox9.(A) A schematic shows that the 5′ end of miR-145 contains a sequence complementary to the specific miRNA binding site within the 3′-UTR of Sox9 mRNA. (B) Pre-miR-145 or its negative control was co-transfected with the specific pMIR-REPORT construct containing a consensus miR-145-binding site (pMIR-PT) into HEK293 cells. Pre-miR-145 or its negative control was co-transfected with pMIR-MRE into HEK293 cells. pMIR-PT acts as a positive control. (C) HEK293 cells were co-transfected with pMIR-MRE together with varying amounts of pre-miR-145, as indicated. Statistical comparisons were made between multiple groups by ANOVA. (D) Anti-miR-145 or its negative control was co-transfected with either pre-miR-145 together with pMIR-MRE or pre-miR-145 together with pMIR-MUT into HEK293 cells. pMIR-PT acts as a positive control. All cells (B, C, D) were harvested at 48 h after transfection, and then luciferase activities were measured and normalized to the phRL-TK activities. Three independent transfection experiments were done and data was represented as mean±sd. *, p<0.05, **, p<0.01, when compared with control.
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pone-0021679-g002: Mir-145 directly targets Sox9.(A) A schematic shows that the 5′ end of miR-145 contains a sequence complementary to the specific miRNA binding site within the 3′-UTR of Sox9 mRNA. (B) Pre-miR-145 or its negative control was co-transfected with the specific pMIR-REPORT construct containing a consensus miR-145-binding site (pMIR-PT) into HEK293 cells. Pre-miR-145 or its negative control was co-transfected with pMIR-MRE into HEK293 cells. pMIR-PT acts as a positive control. (C) HEK293 cells were co-transfected with pMIR-MRE together with varying amounts of pre-miR-145, as indicated. Statistical comparisons were made between multiple groups by ANOVA. (D) Anti-miR-145 or its negative control was co-transfected with either pre-miR-145 together with pMIR-MRE or pre-miR-145 together with pMIR-MUT into HEK293 cells. pMIR-PT acts as a positive control. All cells (B, C, D) were harvested at 48 h after transfection, and then luciferase activities were measured and normalized to the phRL-TK activities. Three independent transfection experiments were done and data was represented as mean±sd. *, p<0.05, **, p<0.01, when compared with control.

Mentions: MiRNAs inhibit mRNA expression by binding the MREs located in 3′-UTR of target mRNA. The Sox9 3′-UTR contains one putative miR-145 seed site which is bound with imperfect complementation (Figure 2A). To determine if miR-145 targets Sox9, we applied the luciferase report gene assay using the pMIR-REPORT Luciferase reporter. Firstly, we constructed a reporter vector containing a consensus miR-145-binding site within the 3′-UTR (pMIR-PT, Table 1) as a positive control. After we co-transfected this reporter plasmid into HEK293 cells with pre-miR-145 or its control pre-miR, we found that luciferase expression significantly decreased in the HEK293 cells transfected with pre-miR-145 (Figure 2B). These data indicate miR-145 can suppress expression of transcripts containing an exact miR-145-binding site by our luciferase reporter assay. Next, we created specific reporter vectors, which contained either two copies of the endogenous MREs found in the Sox9 mRNA 3′-UTR (pMIR-MRE, Table 1) or corresponding two copies of the MREs with a scrambled seed sequence (pMIR-MUT, Table 1). Previous studies have proven that such reporter constructed with multiple MREs is an available method for the identification of miRNAs target genes [37], [38], [39], [40]. Co-transfection of pre-miR-145 with wild-type pMIR-MRE resulted in a suppression of luciferase gene expression, but co-transfection of pre-miR-145 and mutant pMIR-MUT did not (Figure 2B). Furthermore, the suppression effect occurred in a dose dependent manner (Figure 2C). In addition we found that anti-miR-145 could overcome the suppression effect when it was co-transfected with pMIR-MRE and pre-miR-145. However, the effect of anti-miR-145 was abolished when pMIR-MUT was used instead of pMIR-MRE (Figure 2D). Taken together, our data suggest that miR-145 could target Sox9 by binding the MREs within the Sox9 mRNA 3′-UTR.


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 directly targets Sox9.(A) A schematic shows that the 5′ end of miR-145 contains a sequence complementary to the specific miRNA binding site within the 3′-UTR of Sox9 mRNA. (B) Pre-miR-145 or its negative control was co-transfected with the specific pMIR-REPORT construct containing a consensus miR-145-binding site (pMIR-PT) into HEK293 cells. Pre-miR-145 or its negative control was co-transfected with pMIR-MRE into HEK293 cells. pMIR-PT acts as a positive control. (C) HEK293 cells were co-transfected with pMIR-MRE together with varying amounts of pre-miR-145, as indicated. Statistical comparisons were made between multiple groups by ANOVA. (D) Anti-miR-145 or its negative control was co-transfected with either pre-miR-145 together with pMIR-MRE or pre-miR-145 together with pMIR-MUT into HEK293 cells. pMIR-PT acts as a positive control. All cells (B, C, D) were harvested at 48 h after transfection, and then luciferase activities were measured and normalized to the phRL-TK activities. Three independent transfection experiments were done and data was represented as mean±sd. *, p<0.05, **, p<0.01, when compared with control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140487&req=5

pone-0021679-g002: Mir-145 directly targets Sox9.(A) A schematic shows that the 5′ end of miR-145 contains a sequence complementary to the specific miRNA binding site within the 3′-UTR of Sox9 mRNA. (B) Pre-miR-145 or its negative control was co-transfected with the specific pMIR-REPORT construct containing a consensus miR-145-binding site (pMIR-PT) into HEK293 cells. Pre-miR-145 or its negative control was co-transfected with pMIR-MRE into HEK293 cells. pMIR-PT acts as a positive control. (C) HEK293 cells were co-transfected with pMIR-MRE together with varying amounts of pre-miR-145, as indicated. Statistical comparisons were made between multiple groups by ANOVA. (D) Anti-miR-145 or its negative control was co-transfected with either pre-miR-145 together with pMIR-MRE or pre-miR-145 together with pMIR-MUT into HEK293 cells. pMIR-PT acts as a positive control. All cells (B, C, D) were harvested at 48 h after transfection, and then luciferase activities were measured and normalized to the phRL-TK activities. Three independent transfection experiments were done and data was represented as mean±sd. *, p<0.05, **, p<0.01, when compared with control.
Mentions: MiRNAs inhibit mRNA expression by binding the MREs located in 3′-UTR of target mRNA. The Sox9 3′-UTR contains one putative miR-145 seed site which is bound with imperfect complementation (Figure 2A). To determine if miR-145 targets Sox9, we applied the luciferase report gene assay using the pMIR-REPORT Luciferase reporter. Firstly, we constructed a reporter vector containing a consensus miR-145-binding site within the 3′-UTR (pMIR-PT, Table 1) as a positive control. After we co-transfected this reporter plasmid into HEK293 cells with pre-miR-145 or its control pre-miR, we found that luciferase expression significantly decreased in the HEK293 cells transfected with pre-miR-145 (Figure 2B). These data indicate miR-145 can suppress expression of transcripts containing an exact miR-145-binding site by our luciferase reporter assay. Next, we created specific reporter vectors, which contained either two copies of the endogenous MREs found in the Sox9 mRNA 3′-UTR (pMIR-MRE, Table 1) or corresponding two copies of the MREs with a scrambled seed sequence (pMIR-MUT, Table 1). Previous studies have proven that such reporter constructed with multiple MREs is an available method for the identification of miRNAs target genes [37], [38], [39], [40]. Co-transfection of pre-miR-145 with wild-type pMIR-MRE resulted in a suppression of luciferase gene expression, but co-transfection of pre-miR-145 and mutant pMIR-MUT did not (Figure 2B). Furthermore, the suppression effect occurred in a dose dependent manner (Figure 2C). In addition we found that anti-miR-145 could overcome the suppression effect when it was co-transfected with pMIR-MRE and pre-miR-145. However, the effect of anti-miR-145 was abolished when pMIR-MUT was used instead of pMIR-MRE (Figure 2D). Taken together, our data suggest that miR-145 could target Sox9 by binding the MREs within the Sox9 mRNA 3′-UTR.

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