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Thyroid hormone may regulate mRNA abundance in liver by acting on microRNAs.

Dong H, Paquette M, Williams A, Zoeller RT, Wade M, Yauk C - PLoS ONE (2010)

Bottom Line: TH treatment of cells over-expressing miR-206 resulted in decreased miR-206 expression, and a significant increase in two predicted target genes, Mup1 and Gpd2.The results suggest that TH regulation of these genes may occur secondarily via miR-206.These studies provide new insight into the role of miRNAs in mediating TH regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Environmental Health Sciences and Research Bureau, Health Canada, Ottawa, Ontario, Canada. hongyan_dong@hc-sc.gc.ca

ABSTRACT
MicroRNAs (miRNAs) are extensively involved in diverse biological processes. However, very little is known about the role of miRNAs in mediating the action of thyroid hormones (TH). Appropriate TH levels are known to be critically important for development, differentiation and maintenance of metabolic balance in mammals. We induced transient hypothyroidism in juvenile mice by short-term exposure to methimazole and perchlorate from post natal day (PND) 12 to 15. The expression of miRNAs in the liver was analyzed using Taqman Low Density Arrays (containing up to 600 rodent miRNAs). We found the expression of 40 miRNAs was significantly altered in the livers of hypothyroid mice compared to euthyroid controls. Among the miRNAs, miRs-1, 206, 133a and 133b exhibited a massive increase in expression (50- to 500-fold). The regulation of TH on the expression of miRs-1, 206, 133a and 133b was confirmed in various mouse models including: chronic hypothyroid, short-term hyperthyroid and short-term hypothyroid followed by TH supplementation. TH regulation of these miRNAs was also confirmed in mouse hepatocyte AML 12 cells. The expression of precursors of miRs-1, 206, 133a and 133b were examined in AML 12 cells and shown to decrease after TH treatment, only pre-mir-206 and pre-mir-133b reached statistical significance. To identify the targets of these miRNAs, DNA microarrays were used to examine hepatic mRNA levels in the short-term hypothyroid mouse model relative to controls. We found transcripts from 92 known genes were significantly altered in these hypothyroid mice. Web-based target predication software (TargetScan and Microcosm) identified 14 of these transcripts as targets of miRs-1, 206, 133a and 133b. The vast majority of these mRNA targets were significantly down-regulated in hypothyroid mice, corresponding with the up-regulation of miRs-1, 206, 133a and 133b in hypothyroid mouse liver. To further investigate target genes, miR-206 was over-expressed in AML 12 cells. TH treatment of cells over-expressing miR-206 resulted in decreased miR-206 expression, and a significant increase in two predicted target genes, Mup1 and Gpd2. The results suggest that TH regulation of these genes may occur secondarily via miR-206. These studies provide new insight into the role of miRNAs in mediating TH regulation of gene expression.

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Expression of miRs-1, 206, 133a and 133b in other animal models with altered TH levels.A. Hepatic miRNA expression in livers of PND 15 mouse pups rendered hypothyroid by treatment with drinking water containing 0.04% (wt/vol) of PTU from GD 13 to PND 15. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). B. Hepatic miRNA expression in livers of PND 15 mice whose TH levels were modulated as follows: hyperthyroid pups (hyper) received a s.c. injection of T4+T3 (50 µg+5 µg, respectively per 100g bw) four hours prior to sacrifice and corrected hypothyroid pups received drinking water containing MMI and perchlorate (0.05 and 1% wt/vol, respectively) from PND 12 to 15 and an injection of T4+T3 (20 µg+2 µg, respectively per 100g bw) four hours prior to sacrifice, while control mice received an injection of PBS only. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). Data are presented as mean ± SE (n = 3). A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.
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pone-0012136-g002: Expression of miRs-1, 206, 133a and 133b in other animal models with altered TH levels.A. Hepatic miRNA expression in livers of PND 15 mouse pups rendered hypothyroid by treatment with drinking water containing 0.04% (wt/vol) of PTU from GD 13 to PND 15. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). B. Hepatic miRNA expression in livers of PND 15 mice whose TH levels were modulated as follows: hyperthyroid pups (hyper) received a s.c. injection of T4+T3 (50 µg+5 µg, respectively per 100g bw) four hours prior to sacrifice and corrected hypothyroid pups received drinking water containing MMI and perchlorate (0.05 and 1% wt/vol, respectively) from PND 12 to 15 and an injection of T4+T3 (20 µg+2 µg, respectively per 100g bw) four hours prior to sacrifice, while control mice received an injection of PBS only. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). Data are presented as mean ± SE (n = 3). A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.

Mentions: To further investigate the effect of TH on hepatic miRNA expression, we examined the expression of the most differentially regulated miRNAs (miRs-1, 206, 133a, 133b) in the livers of (a) hypothyroid mice induced by PTU treatment (PTU hypothyroid); (b) hyperthyroid mice created by injecting T3/T4 four hours before sacrifice (hyperthyroid) and (c) hypothyroid mice induced by MMI/Perchlorate treatment but receiving T4/T3 injection four hours before sacrifice (corrected hypothyroid). Three mice were chosen from each group and their serum T4 levels were shown in Table 2. As shown in Fig. 2A, all of four selected miRNAs were significantly increased in the livers of PTU induced hypothyroid mice, while significantly decreased in the livers of hyperthyroid mice. Corrected hypothyroid animals had serum T4 levels intermediate between control and hyper thyroid animals although these were only significantly different from the hyperthyroid T4 levels (p = 0.046 vs hyperthyroid and 0.067 vs control; Table 2). Similarly, hepatic expression of all 4 miRNAs was also intermediate between control and hyperthyroid mice with only miR206 being significantly reduced relative to control animals (Fig 2B).


Thyroid hormone may regulate mRNA abundance in liver by acting on microRNAs.

Dong H, Paquette M, Williams A, Zoeller RT, Wade M, Yauk C - PLoS ONE (2010)

Expression of miRs-1, 206, 133a and 133b in other animal models with altered TH levels.A. Hepatic miRNA expression in livers of PND 15 mouse pups rendered hypothyroid by treatment with drinking water containing 0.04% (wt/vol) of PTU from GD 13 to PND 15. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). B. Hepatic miRNA expression in livers of PND 15 mice whose TH levels were modulated as follows: hyperthyroid pups (hyper) received a s.c. injection of T4+T3 (50 µg+5 µg, respectively per 100g bw) four hours prior to sacrifice and corrected hypothyroid pups received drinking water containing MMI and perchlorate (0.05 and 1% wt/vol, respectively) from PND 12 to 15 and an injection of T4+T3 (20 µg+2 µg, respectively per 100g bw) four hours prior to sacrifice, while control mice received an injection of PBS only. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). Data are presented as mean ± SE (n = 3). A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.
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pone-0012136-g002: Expression of miRs-1, 206, 133a and 133b in other animal models with altered TH levels.A. Hepatic miRNA expression in livers of PND 15 mouse pups rendered hypothyroid by treatment with drinking water containing 0.04% (wt/vol) of PTU from GD 13 to PND 15. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). B. Hepatic miRNA expression in livers of PND 15 mice whose TH levels were modulated as follows: hyperthyroid pups (hyper) received a s.c. injection of T4+T3 (50 µg+5 µg, respectively per 100g bw) four hours prior to sacrifice and corrected hypothyroid pups received drinking water containing MMI and perchlorate (0.05 and 1% wt/vol, respectively) from PND 12 to 15 and an injection of T4+T3 (20 µg+2 µg, respectively per 100g bw) four hours prior to sacrifice, while control mice received an injection of PBS only. RT-PCR was performed with the Taqman miRNA Assay with RNA derived from male pups (3 per group). Data are presented as mean ± SE (n = 3). A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.
Mentions: To further investigate the effect of TH on hepatic miRNA expression, we examined the expression of the most differentially regulated miRNAs (miRs-1, 206, 133a, 133b) in the livers of (a) hypothyroid mice induced by PTU treatment (PTU hypothyroid); (b) hyperthyroid mice created by injecting T3/T4 four hours before sacrifice (hyperthyroid) and (c) hypothyroid mice induced by MMI/Perchlorate treatment but receiving T4/T3 injection four hours before sacrifice (corrected hypothyroid). Three mice were chosen from each group and their serum T4 levels were shown in Table 2. As shown in Fig. 2A, all of four selected miRNAs were significantly increased in the livers of PTU induced hypothyroid mice, while significantly decreased in the livers of hyperthyroid mice. Corrected hypothyroid animals had serum T4 levels intermediate between control and hyper thyroid animals although these were only significantly different from the hyperthyroid T4 levels (p = 0.046 vs hyperthyroid and 0.067 vs control; Table 2). Similarly, hepatic expression of all 4 miRNAs was also intermediate between control and hyperthyroid mice with only miR206 being significantly reduced relative to control animals (Fig 2B).

Bottom Line: TH treatment of cells over-expressing miR-206 resulted in decreased miR-206 expression, and a significant increase in two predicted target genes, Mup1 and Gpd2.The results suggest that TH regulation of these genes may occur secondarily via miR-206.These studies provide new insight into the role of miRNAs in mediating TH regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Environmental Health Sciences and Research Bureau, Health Canada, Ottawa, Ontario, Canada. hongyan_dong@hc-sc.gc.ca

ABSTRACT
MicroRNAs (miRNAs) are extensively involved in diverse biological processes. However, very little is known about the role of miRNAs in mediating the action of thyroid hormones (TH). Appropriate TH levels are known to be critically important for development, differentiation and maintenance of metabolic balance in mammals. We induced transient hypothyroidism in juvenile mice by short-term exposure to methimazole and perchlorate from post natal day (PND) 12 to 15. The expression of miRNAs in the liver was analyzed using Taqman Low Density Arrays (containing up to 600 rodent miRNAs). We found the expression of 40 miRNAs was significantly altered in the livers of hypothyroid mice compared to euthyroid controls. Among the miRNAs, miRs-1, 206, 133a and 133b exhibited a massive increase in expression (50- to 500-fold). The regulation of TH on the expression of miRs-1, 206, 133a and 133b was confirmed in various mouse models including: chronic hypothyroid, short-term hyperthyroid and short-term hypothyroid followed by TH supplementation. TH regulation of these miRNAs was also confirmed in mouse hepatocyte AML 12 cells. The expression of precursors of miRs-1, 206, 133a and 133b were examined in AML 12 cells and shown to decrease after TH treatment, only pre-mir-206 and pre-mir-133b reached statistical significance. To identify the targets of these miRNAs, DNA microarrays were used to examine hepatic mRNA levels in the short-term hypothyroid mouse model relative to controls. We found transcripts from 92 known genes were significantly altered in these hypothyroid mice. Web-based target predication software (TargetScan and Microcosm) identified 14 of these transcripts as targets of miRs-1, 206, 133a and 133b. The vast majority of these mRNA targets were significantly down-regulated in hypothyroid mice, corresponding with the up-regulation of miRs-1, 206, 133a and 133b in hypothyroid mouse liver. To further investigate target genes, miR-206 was over-expressed in AML 12 cells. TH treatment of cells over-expressing miR-206 resulted in decreased miR-206 expression, and a significant increase in two predicted target genes, Mup1 and Gpd2. The results suggest that TH regulation of these genes may occur secondarily via miR-206. These studies provide new insight into the role of miRNAs in mediating TH regulation of gene expression.

Show MeSH
Related in: MedlinePlus