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

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.

Figure 1. Serum T4 levels and liver malic enzyme mRNA levels in mouse pups rendered hypothyroid by 3 day exposure of dams and their litters to drinking water containing MMI and perchlorate from PND 12 to 15.

A. Serum T4 data are expressed as mean ± standard error (SE, n = 10) and * indicates significant difference (p<0.001). B. qRT-PCR for malic enzyme mRNA (positively regulated by direct TH action) was performed with RNA derived from male pups. Data are presented as mean ± SE (n = 5). A two-tailed Student's t-test was used to calculate significance and * indicates p = 0.03.

Figure 2. 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.

Figure 3. Expression of miRs-1, 206, 133a and 133b as well as their precursors in the AML 12 cells.

A. AML 12 cells were treated with 10 nm T3 for 1 hour or 24 hours. The expression of miRs-1, 206, 133a and 133b was examined with the Taqman miRNA Assay. U6 was used as an internal control. Five batches of cultured cells were considered as 5 biological replicates. B. The expression of the precursors of miRs-1, 206, 133a and 133b was examined in AML 12 cell treated with 10 nm T3 for 24 hours with RT-PCR. Hprt was used as internal control. Three batches of cultured cells were used as 3 biological replicates. Data are presented as mean ± SE. A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.

Figure 4. Identification of the target genes of miR-206 that are regulated by TH.

A. Levels of miR-206 in AML 12 cells stably transfected to ectopically express miR-206. AML12 cells were transfected with pEGP-mmu-mir-206 expression vector or pEGP-mir null control vector and selected with puromycin and green florescence protein by microscopy. The expression of miR-206 was examined with the Taqman miRNA Assay (n = 5). B. The expression of genes that are putative targets of miR-206 in the two cell types shown in figure 4A was examined with RT-PCR (n = 3). C. The effects of TH on the expression of miR-206 in the transfected cells. The miR-206 transfected cells were treated with 10 nm T3 for 24 hours. MiR-206 expression was analyzed with Taqman miRNA Assay (n = 3). D. The effect of TH on the expression of the miR-206 target genes in the transfected cells. The miR-206 transfected cells were treated with 10 nm T3 for 24 hours. The expression of target genes was examined with RT-PCR (n = 3). Data are presented as mean ± SE. A two-tailed Student's t-test was used to calculate significance. * indicates p<0.05.