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miRNA-mediated functional changes through co-regulating function related genes.

He J, Zhang JF, Yi C, Lv Q, Xie WD, Li JN, Wan G, Cui K, Kung HF, Yang J, Yang BB, Zhang Y - PLoS ONE (2010)

Bottom Line: In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1.With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect.This might also be one of the modes of miRNA-mediated gene regulation.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing, People's Republic of China.

ABSTRACT

Background: MicroRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation.

Results: To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1.

Conclusions: With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.

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miR-20b activates BMP/Runx2 signaling pathway by down-regulating the expression of PPARγ, Bambi and Crim1.A. Predicted (Ai) and mutated (Aii) miR-20b binding sites in the 3′UTR of PPARγ. (Aiii) luciferase activity of the pRL-TK-PPARγ report vector was inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of PPARγ eliminated the repressive effect of miR-20b (Aiv) (n = 6; *p<0.05). p-p+NC: pRL-TK-PPARγ+ negative control; p-p+20b: pRL-TK-PPARγ + miR-20b; p-p+20bI: pRL-TK-PPARγ + miR-20b inhibitor. p-p-mut+NC: pRL-TK-PPARγ mutation plasmid+ negative control; p-p-mut+20b: pRL-TK-PPARγ mutation plasmid+miR-20b; p-p-mut+20bI: pRL-TK-PPARγ mutation plasmid+miR-20b inhibitor. B. Prediction (Bi) and mutation (Bii) of miR-20b binding sites in the 3′UTRs of Bambi. (Biii) Luciferase activities of report vector with the 3′UTR of Bambi were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Bambi (Biv) eliminated the repressive effect of miR-20b (n = 6; *p<0.05). C. Prediction (Ci) and mutation (Cii) of miR-20b binding sites in the 3′UTRs of Crim1. (Ciii) Luciferase activities of report vector with the 3′UTR of Crim1 were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Crim1 (Civ) eliminated the repressive effect of miR-20b (n = 6; **p<0.01). p-B/p-C+NC: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + negative control; p-B/p-C+20b: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b; p-B/p-C+20bI: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b inhibitor. p-B-mut/p-C-mut+NC: pRL-TK-Bambi/pRL-TK-Crim1mutation plasmid + negative control; p-B-mut/p-C-mut+20b: pRL-TK-Bambi /pRL-TK- Crim1 mutation plasmid + miR-20b; p-B-mut/p-C-mut+20bI: pRL-TK-Bambi/pRL-TK- Crim1 mutation plasmid + miR-20b inhibitor.
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pone-0013558-g003: miR-20b activates BMP/Runx2 signaling pathway by down-regulating the expression of PPARγ, Bambi and Crim1.A. Predicted (Ai) and mutated (Aii) miR-20b binding sites in the 3′UTR of PPARγ. (Aiii) luciferase activity of the pRL-TK-PPARγ report vector was inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of PPARγ eliminated the repressive effect of miR-20b (Aiv) (n = 6; *p<0.05). p-p+NC: pRL-TK-PPARγ+ negative control; p-p+20b: pRL-TK-PPARγ + miR-20b; p-p+20bI: pRL-TK-PPARγ + miR-20b inhibitor. p-p-mut+NC: pRL-TK-PPARγ mutation plasmid+ negative control; p-p-mut+20b: pRL-TK-PPARγ mutation plasmid+miR-20b; p-p-mut+20bI: pRL-TK-PPARγ mutation plasmid+miR-20b inhibitor. B. Prediction (Bi) and mutation (Bii) of miR-20b binding sites in the 3′UTRs of Bambi. (Biii) Luciferase activities of report vector with the 3′UTR of Bambi were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Bambi (Biv) eliminated the repressive effect of miR-20b (n = 6; *p<0.05). C. Prediction (Ci) and mutation (Cii) of miR-20b binding sites in the 3′UTRs of Crim1. (Ciii) Luciferase activities of report vector with the 3′UTR of Crim1 were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Crim1 (Civ) eliminated the repressive effect of miR-20b (n = 6; **p<0.01). p-B/p-C+NC: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + negative control; p-B/p-C+20b: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b; p-B/p-C+20bI: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b inhibitor. p-B-mut/p-C-mut+NC: pRL-TK-Bambi/pRL-TK-Crim1mutation plasmid + negative control; p-B-mut/p-C-mut+20b: pRL-TK-Bambi /pRL-TK- Crim1 mutation plasmid + miR-20b; p-B-mut/p-C-mut+20bI: pRL-TK-Bambi/pRL-TK- Crim1 mutation plasmid + miR-20b inhibitor.

Mentions: We did so by using FindTar, a computational algorithm developed in our Lab (http://bio.sz.tsinghua.edu.cn/lab/findtar) [31], and found that miR-20b consisted of a highly probable binding site at the 3′UTR of PPARγ (Fig. 3Ai). We then further investigated the interaction between the 3′UTR of PPARγ and miR-20b. The 3′UTR of PPARγ was inserted into a luciferase expression vector to generate a luciferase reporter construct. The miR-20b was then co-transfected with this reporter construct into Cos-7 cells and the levels of luciferase activity were measured to determine the corresponding repressive effects. Indeed, we detected a significant repression in luciferase activity when miR-20b was co-transfected with this vector expressing the 3′UTR of PPARγ (Fig. 3Aii). To further confirm this direct interaction, the binding site in PPARγ 3′UTR was mutated to generate another reporter vector (Fig. 3Aiii). Such a mutation abolished the repressive effects that miR-20b had on luciferase activity (Fig. 3Ai), suggesting that miR-20b regulates PPARγ expression by directly binding to the 3′UTR site of PPARγ.


miRNA-mediated functional changes through co-regulating function related genes.

He J, Zhang JF, Yi C, Lv Q, Xie WD, Li JN, Wan G, Cui K, Kung HF, Yang J, Yang BB, Zhang Y - PLoS ONE (2010)

miR-20b activates BMP/Runx2 signaling pathway by down-regulating the expression of PPARγ, Bambi and Crim1.A. Predicted (Ai) and mutated (Aii) miR-20b binding sites in the 3′UTR of PPARγ. (Aiii) luciferase activity of the pRL-TK-PPARγ report vector was inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of PPARγ eliminated the repressive effect of miR-20b (Aiv) (n = 6; *p<0.05). p-p+NC: pRL-TK-PPARγ+ negative control; p-p+20b: pRL-TK-PPARγ + miR-20b; p-p+20bI: pRL-TK-PPARγ + miR-20b inhibitor. p-p-mut+NC: pRL-TK-PPARγ mutation plasmid+ negative control; p-p-mut+20b: pRL-TK-PPARγ mutation plasmid+miR-20b; p-p-mut+20bI: pRL-TK-PPARγ mutation plasmid+miR-20b inhibitor. B. Prediction (Bi) and mutation (Bii) of miR-20b binding sites in the 3′UTRs of Bambi. (Biii) Luciferase activities of report vector with the 3′UTR of Bambi were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Bambi (Biv) eliminated the repressive effect of miR-20b (n = 6; *p<0.05). C. Prediction (Ci) and mutation (Cii) of miR-20b binding sites in the 3′UTRs of Crim1. (Ciii) Luciferase activities of report vector with the 3′UTR of Crim1 were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Crim1 (Civ) eliminated the repressive effect of miR-20b (n = 6; **p<0.01). p-B/p-C+NC: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + negative control; p-B/p-C+20b: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b; p-B/p-C+20bI: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b inhibitor. p-B-mut/p-C-mut+NC: pRL-TK-Bambi/pRL-TK-Crim1mutation plasmid + negative control; p-B-mut/p-C-mut+20b: pRL-TK-Bambi /pRL-TK- Crim1 mutation plasmid + miR-20b; p-B-mut/p-C-mut+20bI: pRL-TK-Bambi/pRL-TK- Crim1 mutation plasmid + miR-20b inhibitor.
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getmorefigures.php?uid=PMC2962631&req=5

pone-0013558-g003: miR-20b activates BMP/Runx2 signaling pathway by down-regulating the expression of PPARγ, Bambi and Crim1.A. Predicted (Ai) and mutated (Aii) miR-20b binding sites in the 3′UTR of PPARγ. (Aiii) luciferase activity of the pRL-TK-PPARγ report vector was inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of PPARγ eliminated the repressive effect of miR-20b (Aiv) (n = 6; *p<0.05). p-p+NC: pRL-TK-PPARγ+ negative control; p-p+20b: pRL-TK-PPARγ + miR-20b; p-p+20bI: pRL-TK-PPARγ + miR-20b inhibitor. p-p-mut+NC: pRL-TK-PPARγ mutation plasmid+ negative control; p-p-mut+20b: pRL-TK-PPARγ mutation plasmid+miR-20b; p-p-mut+20bI: pRL-TK-PPARγ mutation plasmid+miR-20b inhibitor. B. Prediction (Bi) and mutation (Bii) of miR-20b binding sites in the 3′UTRs of Bambi. (Biii) Luciferase activities of report vector with the 3′UTR of Bambi were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Bambi (Biv) eliminated the repressive effect of miR-20b (n = 6; *p<0.05). C. Prediction (Ci) and mutation (Cii) of miR-20b binding sites in the 3′UTRs of Crim1. (Ciii) Luciferase activities of report vector with the 3′UTR of Crim1 were inhibited by miR-20b and the mutation on the binding sites of the 3′UTR of Crim1 (Civ) eliminated the repressive effect of miR-20b (n = 6; **p<0.01). p-B/p-C+NC: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + negative control; p-B/p-C+20b: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b; p-B/p-C+20bI: pRL-TK-Bambi/pRL-TK-Crim1 plasmid + miR-20b inhibitor. p-B-mut/p-C-mut+NC: pRL-TK-Bambi/pRL-TK-Crim1mutation plasmid + negative control; p-B-mut/p-C-mut+20b: pRL-TK-Bambi /pRL-TK- Crim1 mutation plasmid + miR-20b; p-B-mut/p-C-mut+20bI: pRL-TK-Bambi/pRL-TK- Crim1 mutation plasmid + miR-20b inhibitor.
Mentions: We did so by using FindTar, a computational algorithm developed in our Lab (http://bio.sz.tsinghua.edu.cn/lab/findtar) [31], and found that miR-20b consisted of a highly probable binding site at the 3′UTR of PPARγ (Fig. 3Ai). We then further investigated the interaction between the 3′UTR of PPARγ and miR-20b. The 3′UTR of PPARγ was inserted into a luciferase expression vector to generate a luciferase reporter construct. The miR-20b was then co-transfected with this reporter construct into Cos-7 cells and the levels of luciferase activity were measured to determine the corresponding repressive effects. Indeed, we detected a significant repression in luciferase activity when miR-20b was co-transfected with this vector expressing the 3′UTR of PPARγ (Fig. 3Aii). To further confirm this direct interaction, the binding site in PPARγ 3′UTR was mutated to generate another reporter vector (Fig. 3Aiii). Such a mutation abolished the repressive effects that miR-20b had on luciferase activity (Fig. 3Ai), suggesting that miR-20b regulates PPARγ expression by directly binding to the 3′UTR site of PPARγ.

Bottom Line: In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1.With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect.This might also be one of the modes of miRNA-mediated gene regulation.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing, People's Republic of China.

ABSTRACT

Background: MicroRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation.

Results: To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1.

Conclusions: With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.

Show MeSH
Related in: MedlinePlus