ORIGINAL

RESEARCH

Thyroglobulin Increases Thyroid Cell Proliferation via the Suppression of Specific MicroRNAs
Takeshi Akama, Yuqian Luo, Donald F. Sellitti, Akira Kawashima, Kazunari Tanigawa, Aya Yoshihara, Yuko Ishido, Kazuaki Nakamura, Akito Tanoue, and Koichi Suzuki
Laboratory of Molecular Diagnostics (T.A., Y.L., A.K., K.T., A.Y, Y.I., K.N., K.S.), Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Higashimurayamashi, Tokyo 189 0002, Japan; Department of Medicine, Uniformed Services University of the Health Sciences (D.F.S.), Bethesda, Maryland 20814 4799; and Department of Pharmacology, National Research Institute for Child Health and Development (K.N., A.T.), Setagaya-ku, Tokyo 157 8538, Japan Thyroglobulin (Tg), stored in the follicular lumen, has also been shown recently to perform two unexpected roles: as an autocrine negative-feedback suppressor of thyroid function in the presence of TSH and as a potent inducer of thyroid cell growth in the absence of TSH. However, the underlying molecular mechanism(s) remain unclear. To elucidate a molecular pathway linking Tg to increased cell proliferation, we examined the regulation of microRNAs (miRNAs) by Tg using an miRNA microarray. We identified 21 miRNAs whose expression was significantly suppressed by Tg in rat thyroid FRTL-5 cells. Using specific miRNA analogs, we determined that miR-16, miR-24, and miR-195 mediate the induction of thyroid cell growth by Tg. The expression of miR-16 and miR-195 target genes, Mapk8, Ccne1, and Cdc6, which were previously shown to be essential for TSHstimulated thyroid cell growth, were also induced by Tg. Moreover, the Tg-induced expression of these genes was reduced by overexpression of miR-16 and miR-195. Similarly, the induction of c-Myc by Tg was reduced by miR-24 overexpression. These results suggest that Tg could alter thyroid cell proliferation by increasing the expression of cell division-related genes such as Mapk8, Ccne1, Cdc6, and c-Myc through its suppression of specific microRNAs (miR-16, miR-24, and miR195). In addition, we identified phosphatidylinositol 3-kinase as a key signaling pathway, linking Tg with cell proliferation. The present data support an important role for miRNAs as effectors for the effect of Tg on cell proliferation and perhaps other functions of Tg in the thyroid cell. (Molecular Endocrinology 28: 368 379, 2014)

hyroglobulin (Tg) is a major protein product of the thyroid that serves as the precursor for thyroid hormone formation (1). The protein is translated into a 330kDa monomer and glycosylated at specific residues within the thyroid follicular cell and then secreted as a dimer into the follicular lumen in which specific tyrosyl residues are iodinated and coupled to form intramolecular T4 and T3. The thyroid hormones, in turn, are released into the circulation after the endocytosis and lysosomal digestion of hormone-laden Tg (1). This process is not synchronized but rather occurs seemingly at random throughout the thyroid, creating the characteristic heter-

ogeneity of Tg content, size, and cellular morphology in the thyroid follicle, the minimal functional unit of the thyroid gland. Thus, although some follicles are actively engaged in iodide transport, organification, and covalent coupling to produce new thyroid hormone precursor, others, already filled with Tg-bound thyroid hormone serve as storage depots for subsequent endocytosis, lysosomal degradation, and release of free hormone into the blood stream (25). In addition to its canonical role as the scaffold for thyroid hormone synthesis, Tg, in a role only recently discovered, acts as a potent regulator of thyroid function
Abbreviations: BrdU, bromodeoxyuridine; Cdc6, cell division cycle 6; CDK, cyclin-dependent kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; miRNA, microRNA; pax-8, paired box 8; PBST, PBS with 0.1% Tween 20; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; Tg, thyroglobulin; TSHR, TSH receptor; UTR, untranslated region.

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright 2014 by the Endocrine Society Received September 3, 2013. Accepted January 22, 2014. First Published Online January 30, 2014

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by modulating thyroid-specific gene expression (6). Specifically, Tg has been shown to suppress the expression of thyroperoxidase and Tg itself through a reduction in the expression of thyroid transcription factor-1 (Nkx21), thyroid transcription factor-2 (Foxe1), and paired box 8 (Pax8) (6). The expression of the sodium iodide symporter (Slc5a5) and Pendred syndrome gene (Slc26a4), iodide transporters located in the basolateral and apical membranes of the thyroid cell, respectively, are also coordinately regulated by the cyclical variations of colloid Tg content (79). Thus, the long-recognized pattern of morphological and functional heterogeneity of thyroid follicles can best be explained as a reflection of the autoregulation of thyroid function by fluctuating concentrations of follicular Tg. It has been shown, moreover, that Tg increases cell proliferation (10, 11). In early investigations, increased cell proliferation in response to Tg was identified in cultured renal mesangial cells. In these studies, cells exposed to Tg concentrations exceeding those in serum, yet far below those found in normal thyroid follicles (12, 13), exhibited significantly increased growth rates compared with control, whereas T3 and known ligands for Tg-binding proteins had no effect (11). We subsequently reported that Tg acts independently of TSH, insulin, and IGF stimulation to increase proliferation in FRTL-5 cells, a line of hormone-responsive cells derived from rat thyroid (10). Physiological concentrations of Tg (ie, Tg concentrations present in the lumens of thyroid follicles) were shown by us to stimulate FRTL-5 cell proliferation by activation of two intracellular signaling paths, the phosphatidylinositol 3-kinase (PI3K)/Akt pathways that Tg shares with TSH, and a mechanism using c-Raf/MAPK/ERK kinase (MEK)/ERK signaling that appears to be unique to Tg (10, 14). The Tg-recognition system or systems existing upstream of these intracellular signal pathways remain largely unknown at the molecular level, but whatever their nature, their specificity for Tg has received abundant support, including findings that Tg retains its regulatory activity, even after purification using Sephacryl/highpressure gel permeation chromatography (10, 15), and that Tg actions in the thyroid cell can be suppressed using a specific antibody against Tg (10, 16). Moreover, the actions of Tg are not mimicked by factors such as thyroid hormones, iodide, TSH, osmotic pressure, nonspecific protein concentrations, or any other known hormones and cytokines (6, 10, 11, 15, 16). MicroRNAs (miRNAs) are short (23 nucleotides) noncoding RNA sequences transcribed from areas of the genome that had not previously been identified with a definitive function but are now known to have important

actions in cell regulation. Each of these miRNAs, in turn, binds to multiple target mRNAs and down-regulates their translation into active proteins (17). To date, miRNAs have been identified as playing significant roles in cell proliferation, differentiation, and apoptosis (18 22), especially as to their involvement in regulating the growth of certain cancers (23, 24), including papillary (25), follicular (24), and anaplastic (26) cancers of the thyroid. With regard to miRNA function in normal thyroid cell replication, we recently demonstrated, using FRTL-5 thyroid cells, that the familiar role of TSH in increasing thyroid cell proliferation may be due, in part, to its suppression of miR-16 and miR-195 expression, resulting in the increased expression of a group of cell cycle-related target genes that act together to maintain cells in a dividing state (27). In the present study, we extended these studies of miRNA control of thyroid growth to the cell proliferation induced by Tg. Our results have identified several miRNAs (miR-16 and miR-195) and several gene targets of those miRNAs that respond to both TSH and Tg stimulation in FRTL-5 cells. In addition, we have identified an additional miRNA, miR-24, responsible for Myc up-regulation that had previously shown no involvement in the regulation of TSH-induced proliferation but in the present study revealed a significant role in thyroid cell division induced by Tg. We also determined the target genes and signaling cascades responsible for the expression and function of such miRNAs.

Materials and Methods

Culture and treatment of cells
Rat FRTL-5 thyroid cells (ATCC CRL8305) were provided by the Interthyr Research Foundation (Woodinville, WA) and maintained as reported previously (27, 28). The cells were shifted to control medium containing 0.2% serum, but no TSH and insulin, for 7 days before each experiment to induce a quiescent state. Bovine thyroglobulin (Sigma) was added at 5 mg/mL for the indicated period of time. To overexpress or suppress miRNAs, 50 nM of pre-miR miRNA precursor molecule or anti-miR miRNA inhibitor (Life Technologies), respectively, was transfected into quiescent cells using Lipofectamine RNAiMAX (Life Technologies) as described (27). PD98059 (50 M), LY294002 (50 M), or their solvent dimethyl sulfoxide, all from Sigma, were added to the cells beginning 30 minutes prior to Tg treatment.

miRNA microarray
Quiescent FRTL-5 cells were cultured with or without Tg for 24 hours, and then total RNA was extracted with TRIzol solution (Invitrogen) according to the manufacturers recommendation. Small RNA was enriched from 7.5 g of total RNA by using the PureLink miRNA isolation kit (Invitrogen). Fluores-

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cence-labeled RNA was used in an NCode multispecies miRNA V2 microarray (Invitrogen) after Alexafluor labeling as described (27).

(GE Healthcare) and exposure to Amercham Hyperfilm MP (GE Healthcare).

RNA isolation, RT-PCR, and quantitative real-time PCR

Total RNA was purified using an RNeasy Plus minikit (QIAGEN) and used to synthesize cDNA using either a high-capacity cDNA reverse transcription kit (Applied Biosystems) or a TaqMan miRNA reverse transcription kit (Applied Biosystems) as described previously (29). Touchdown PCR was performed using a PCR thermal cycler DICE (TaKaRa Bio) as described (29). Real-time PCR was carried out with 2 ng cDNA, TaqMan miRNA assays (Applied Biosystems), and TaqMan universal PCR master mix (Applied Biosystems) on an ABI Prism 7000 detection system (Applied Biosystems). miRNA expression was normalized to -actin using the threshold cycle method. The primer sequences for rat Mapk8, Ccne1, Cdc6, Myc, and Actb were described previously (14, 27).

Construction of luciferase expression reporter plasmids and luciferase assays

Synthetic oligodeoxynucleotides of the miR-24 binding sites predicted in the Myc 3-untranslated region (UTR) were cloned into miRNA expression reporter vector pMIR-REPORT luciferase (Applied Biosystems) as described previously (27). Reporter gene constructs for Mapk8, Ccne1, and Cdc6 were synthesized and used as described (27). Quiescent FRTL-5 cells cultured in six-well plates were transfected with the corresponding pre-miR miRNA precursor molecule for each target gene, and luciferase activity was measured as described in the previous report (27).

Results
Detection of a subset of miRNAs suppressed by Tg and involved in the regulation of thyroid cell proliferation We first investigated the Tg-responsive miRNA expression profile in FRTL-5 thyroid cells using a miRNA microarray on which 237 rat miRNAs had been mounted in triplicate. Small RNAs prepared from quiescent FRTL-5 cells treated with or without 5 mg/mL Tg for 24 hours were hybridized with the miRNA microarray. Results showed that most miRNAs expressed at mid- to high levels in untreated FRTL-5 cells were reduced by Tg treatment, as indicated by the rightward shift from a theoretical linear correlation between Tg-treated and -untreated cells shown in Figure 1A. When the most Tg-responsive miRNAs were further analyzed using the more quantitative technique of real-time PCR (Figure 1B), we found a total of 21 mRNAs that were reduced by more than 50% from control value by exposure to Tg (Table 1). Of these 21 miRNAs, 19 were the same as those previously identified by us as miRNAs exhibiting a significant decrease with TSH treatment (27). These results suggested the existence of a subset of miRNAs that are common to both TSH- and Tg-induced increases in thyroid cell proliferation, despite the presence of major differences in downstream signaling between the two thyroid-regulatory proteins. For example, the Tg signal for increased cell growth does not involve an up-regulation of intracellular cAMP, whereas the generation of cAMP is important for TSHinduced thyroid cell proliferation (10). Furthermore, Tg treatment significantly reduced mRNA levels of Tshr (Supplemental Figure 1). These results suggest that despite inducing similar changes in miRNA expression, the TSH receptor (TSHR)/cAMP system was not used by Tg to modulate the expression of miRNAs.

Bromodeoxyuridine (BrdU) cell proliferation assay

Quiescent cells inoculated into 96-well plates at a density of 3 103 cells/well were transfected with either pre-miR miRNA precursor molecules or anti-miR miRNA inhibitors using 0.3 L Lipofectamine RNAiMAX (Life Technologies). Cells were then incubated for 30 hours, with BrdU added during the last 6 hours. BrdU incorporation was determined using a 5-bromo2-deoxyuridine labeling and detection kit III (Roche Diagnostics) according to the manufacturers recommendations.

Prediction of miRNA target genes and sequences

Prediction of miRNA target genes was performed using the following programs: TargetScan 6.0 (http://www.targetscan.org/) (30); miRDB (http://mirdb.org/miRDB/) (31); microT 3.0 (http://diana.cslab.ece.ntua.gr/microT/) (32); and miRNA.org (http://www.microrna.org/microrna/home.do) (33).

Protein preparation and Western blot analysis

To extract protein from FRTL-5 cells, cultures were lysed in a buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, and 20% glycerol, followed by the addition of protease inhibitor cocktail tablets (Complete Mini; Roche Diagnostics). Western blot analysis was performed as described (27, 34, 35). Briefly, the proteins were separated on NuPage 4%12% Bis Tris gels by electrophoresis and transferred to i-Blot Gel nitrocellulose transfer stacks (Invitrogen). The membrane was washed with PBS with 0.1% Tween 20 (PBST), placed in blocking buffer (PBST containing 5% nonfat milk) for 1 hour, and then incubated with anticyclin E (Santa Cruz Biotechnology) (1:1000), anti-c-Jun N-terminal kinase (JNK) (BD Biosciences) (1:1000), anti-c-Myc (Cell Signaling) (1:1000), or anti--actin (Santa Cruz Biotechnology) (1:1000) antibody overnight. After washing with PBST, the membrane was incubated for 1 hour with biotinylated antirabbit IgG antibody (GE Healthcare), antimouse IgG antibody (GE Healthcare), or antigoat IgG antibody (Millipore), depending on the species of origin of the primary antibody. After washing with PBST, the membrane was incubated for 1 hour with streptavidin-horseradish peroxidase (GE Healthcare) (1:20000). Horseradish peroxidase was visualized using the ECL Prime reagent

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(10n) 5

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Figure 1. Identification of miRNAs responsible for Tg-induced thyroid cell growth. A, miRNA microarray analysis was performed using quiescent FRTL-5 cells treated with Tg for 24 hours. The signal intensities of the triplicate 237 rat miRNA were plotted. The linear fitted curve is y 0.3439x 40.627, R2 0.8594. B, The same RNA samples used for the microarray were subjected to real-time PCR analysis by TaqMan miRNA assays to evaluate expression of eight representative miRNAs that showed various levels of expression in microarray analysis. The fold decrease of Tg-treated samples was illustrated as the mean SEM (n 3). C, Quiescent FRTL-5 cells were transfected with indicated pre-miR miRNA agonists followed by Tg treatment for 24 hours. BrdU labeling was carried out for 6 hours at the end of incubation. Representative results are shown. The relative amounts of BrdU incorporated were normalized to control samples without Tg treatment and were expressed as the mean SEM (n 6). D, Quiescent FRTL-5 cells grown in six-well plates were transfected with indicated pre-miR miRNA agonists followed by Tg treatment for 48 hours. Cell numbers per well were counted and expressed as the mean SEM (n 3). E, Quiescent FRTL-5 cells were transfected with indicated anti-miR miRNA antagonists and cultured for 48 hours without Tg treatment. BrdU labeling was carried out for 24 hours at the end of incubation. The amounts of BrdU incorporation were normalized to control samples without Tg treatment and were expressed as the mean SEM (n 6). *, P .05, **, P .01 relative to the control levels with Tg treatment by Students t test.

To investigate the function of these miRNAs in Tginduced thyroid cell growth, specific agonists of the precursor for each of these miRNAs (pre-miRs) were transfected into FRTL-5 cells to compensate for their decreased expression in the presence of Tg, and cell proliferation was measured by BrdU incorporation. Our results revealed that only the overexpression of miR-16, miR-24, and miR-195 significantly reduced the increase in BrdU incorporation induced by Tg (Figure 1C). Consistent with their effects on BrdU incorporation, miR-16, miR-195, and miR-24 also significantly reduced total cell number 48 hours after Tg treatment compared with nontransfected controls (Figure 1D). None of the other premiRs tested had a demonstrable effect on Tg-stimulated cell division. This finding was of particular interest be-

cause in a previous study we had shown that two of these three miRNAs (miR-16 and miR-195) were also involved in thyroid cell proliferation induced by TSH. This would suggest that miR-16 and miR-195 are not specific for Tg-induced growth but instead may act more generally to increase cell division in response to a number of upstream agonists, including TSH (27). In contrast, miR-24 had not previously been shown to exert an effect on TSH-induced cell division in FRTL-5 cells (27) and therefore could represent a regulatory element in Tg-stimulated cell proliferation that is distinct from any in the TSH-stimulated pathway. As another demonstration of the involvement of these three miRNAs in Tg-stimulated thyroid cell growth, we suppressed the endogenous expression of miR-16, miR-

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Table 1. miRNAs Decreased More Than Twice by Tg Treatment

Signal Intensity miRNA miR-30b miR-22 miR-16 miR-99b miR-195 miR-30c miR-361 miR-24 miR-320 miR-342 miR-10b miR-22* miR-221 miR-26a miR-130a miR-335 let-7a miR-23a miR-185 miR-27b miR-103
a

Tg () 2139 4875 4985 2297 4750 5568 833 12 045 968 451 155 12 383 5841 28 493 1139 319 30 535 10 301 1100 3798 1683

Tg () 483 1669 1732 801 1667 2057 308 4644 405 190 66 5369 2566 12 539 510 145 13 997 4734 506 1772 792

Fold Change 4.43 2.92 2.88 2.87 2.85 2.71 2.71 2.59 2.39 2.38 2.34 2.31 2.28 2.27 2.23 2.20 2.18 2.18 2.17 2.14 2.13

Decreased by TSHa Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes

Results from previous report (27).

195, and miR-24 by transfecting specific antagonists (anti-miRs) of each miRNA species into quiescent, untreated FRTL-5 cells and subsequently measured BrdU incorporation (Figure 1E). Specific antagonists of both miR-16 and miR-195 significantly increased BrdU incorporation; however, a specific antagonist for miRNA-24 did not significantly affect the incorporation of the labeled nucleotide into DNA (Figure 1E). The cell cycle-related genes, Mapk8, Ccne1, and Cdc6, common targets for miR-16 and miR-195, are regulated by Tg Transcript levels of miR-16, miR-195, and miR-24 exhibited almost identical time-dependent reductions in response to Tg, reaching significance at 3 hours, and declining to approximately 20%30% of control value at 18 hours of Tg treatment (Figure 2A), demonstrating that the suppressive effects of Tg on these miRNAs are persistent in the face of continuous exposure to the protein. To identify possible target genes for these three miRNAs, we reanalyzed the data from a DNA microarray that we had previously performed in quiescent FRTL-5 cells treated with Tg for 24 hours, making the reasonable assumption that miRNA expression levels remained suppressed at least through that time (34). Using four prediction programs (TargetScan, miRDB, microT, and miRNA.org to identify target genes for miRNAs as described in Materi-

als and Methods, we identified 99 genes [of a total of 1166 genes increased at least 2-fold by Tg (34)] that were predicted by at least one of the programs as potential target genes for the three miRNAs. These genes included 70 targets for miR-16, 67 targets for miR-195, and 39 targets for miR-24 (Supplemental Table 1). Among these 99 genes were three potential target genes, Mapk8, Ccne1, and cell division cycle 6 (Cdc6) that we had previously identified as targets of both miR-16 and miR-195 and whose suppression by TSH contributed to TSH-induced thyroid cell proliferation (27). These genes encode, respectively, a MAPK JNK, a cell cycle-related cyclin E, and a component of DNA prereplicative complex Cdc6, each of which functions in progression through the cell cycle. All three genes also possess nearly overlapping target sequences for miR-16 and miR195 in their 3UTRs, probably reflecting the fact that the two miRNAs belong to the same miRNA family and share a high degree of sequence similarity (36). None of these three genes, however, was included among the 39 predicted gene targets for miR-24, nor was miR-24 shown previously to play a role in the TSH-induced cell proliferation (27). This suggests that miR-24, unlike miR-16 and miR-195, has distinct target genes that are specific to Tg-induced, but not to TSH-induced, thyroid cell growth. To examine whether the up-regulation of Mapk8, Ccne1, and Cdc6 by Tg was actually induced by a decrease in miR-16 and/or miR-195 as theoretically predicted, we transfected the agonists of miR-16, miR-195, and miR-24 into Tg-treated cells and observed the effect of their overexpression on the transcript levels of the cell cycle-related genes using conventional RT-PCR (Figure 2B) and real-time PCR (Figure 2C). The results clearly showed that transfection of miR-16 and miR-195 agonists reversed Tg-induced up-regulation of Mapk8, Ccne1, and Cdc6 expression as detected by either PCR method (Figure 2, B and C, respectively). Similarly, levels of the corresponding protein products for these genes (JNK, cyclin E, and Cdc6, respectively), were also suppressed by the agonists of miR-16 and miR-195 in cells treated with Tg (Figure 2, D and E). On the contrary, transfection of the Tg-treated cells with an miR-24 agonist had no effect on either the mRNA expression of the three genes or on the levels of their respective protein product, in agreement with the computer-based prediction of likely miR-24 gene targets. Identification of c-myc as a target for Tg-regulated miR-24 Because no genes directly involved in cell cycle regulation were identified among the predicted target genes for miR-24 (Supplemental Table 1), we investigated the pos-

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Myc encodes a transcription factor MYC that induces the expression 1.0 of series of genes related to cell miR-16 miR-195 growth (37), and MYC has been re0.8 miR-24 ported to be a target for miR-24 in a number of human cell lines (38). To 0.6 Mapk8 confirm our in silico prediction and 0.4 Ccne1 examine whether c-Myc is actually targeted by miR-24 in FRTL-5 cells, Cdc6 0.2 we transfected the cells with agoActb nists (pre-miRs) of miR-24, mR-16, 0 and mR-195 or control and mea0 1 3 6 12 18 h Time after Tg treatment sured relative Myc mRNA levels afD C ter Tg treatment using both conven5 Tg (+) Mapk8 Ccne1 tional RT-PCR (Figure 3B) and real4 Cdc6 time PCR (Figure 3C). Both mRNA 3 quantitation methods demonstrated that overexpression of miR-24 sup 2 pressed the Tg-induced increase in JNK 1 Myc mRNA levels (Figure 3, B and Cyclin E 0 C, respectively). In agreement with Cdc6 the changes in mRNA levels, WestActin ern blot analysis revealed that c-Myc protein was significantly increased by Tg but that this increase was supTg (+) pressed by the overexpression of Figure 2. Mapk8, Ccne1, and Cd6 are target genes for Tg-regulated miR-16 and miR-195. A, miR-24 (Figure 3, D and E). In conQuiescent FRTL-5 cells were treated with Tg for the indicated period of time, and the expression levels of miRNAs were evaluated by real-time PCR. Expression of miRNAs was normalized to trast, agonists of miR-16 and miRcontrol samples without Tg treatment and were expressed as the mean SEM (n 6). B and C, 195 had no effect on either the Quiescent FRTL-5 cells were transfected with indicated pre-miR miRNA agonists followed by Tg mRNA or protein levels of Myc (Figtreatment for 24 hours. Expression of target genes Mapk8, Ccne1, and Cd6 for miR16 and miR195 was evaluated by RT-PCR (B) and real-time PCR analysis (C). mRNA levels were ure 3, BE). normalized to control samples without Tg treatment and were expressed as the mean SEM To ascertain whether the three (n 3). D, Cells were treated the same condition as in B and C, and protein levels were miRNA species control specific gene evaluated by Western blotting. *, P .05, **, P .01 relative to the control levels by Students t test. expression through direct interaction with and cleavage of specific sibility that miR-24 could transduce the increase in cell sites within the 3UTR of their respective target genes, we proliferation by Tg through a less direct means. In a pre- performed luciferase assays using reporter plasmids posvious report, we showed that Tg activates the c-Raf/MEK/ sessing the predicted miRNA binding sequences of the ERK pathway to induce expression of Myc, Fos, and Jun three miRNAs (Supplemental Figure 2). Assays for and that in turn promotes DNA synthesis through the Mapk8, Ccne1, and Cdc6 were as described (27), except activation of specific cyclins and cyclin-dependent kinases that cells in the current study were assayed in a quiescent (CDKs) and p53 (14). Because the mRNA levels of Myc, state. As we had observed previously, luciferase activities Fos and Jun were significantly increased by Tg in 1 hour of Makk8, Ccne1, and Cdc6 were significantly reduced but then declined to original levels by 24 hours (14), it is when miR-16 or miR-195 agonists were transfected, repossible that Tg-induced changes in the expression of iterating the earlier finding that these two miRNAs conthese genes might have eluded detection in our original trol cycle-related genes by direct 3UTR binding. Because DNA microarray analysis of cells 24 hours after Tg treat- Tg also appears to control cell proliferation through ment. We therefore used prediction programs to assess miR-24 and Myc, we generated a luciferase reporter gene whether Myc, Fos, or Jun could constitute target genes for construct possessing a miR-24 recognition sequence and miR-24 and found that only Myc is a high-probability tested the luciferase response to miR-24 agonist (Suppletarget, binding to positions 360 381 in the 3UTR of the mental Figure 2). The results indicate that miR-24 acts by gene (Figure 3A). binding to and cleaving target sequences within the

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A B

Myc 3 UTR(360-381) 5 uggaaaaaauauAAUUGAGCCa 3 ||:|||||| miR-24 3 gacaaggacgacUUGACUCGGu 5

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Figure 3. Identification of c-myc as a target gene for Tg-regulated miR-24. A, The predicted binding sites of miR-24 within the 3UTR (360 381) of rat Myc (accession number NM_012603). A-U and G-C pairs are shown as vertical lines, and G-U is shown as a colon. B and C, Quiescent FRTL5 cells were transfected with indicated pre-miR miRNA agonists followed by Tg treatment for 12 hours. The Myc mRNA level was evaluated by RTPCR (B) and real-time PCR analysis (C). mRNA levels were normalized to control samples without Tg treatment and were expressed as mean SEM (n 3). D and E, Quiescent FRTL-5 cells were transfected with indicated pre-miR miRNA agonists followed by Tg treatment for 24 hours, and the protein level of c-myc was evaluated by Western blotting in three independent experiments. Representative Western blots of c-myc and -actin are shown in panel D. Specific bands on the nitrocellulose membrane were quantified using ImageJ64 software (National Institutes of Health, Bethesda, Maryland). The c-myc protein levels were normalized to the corresponding -actin levels and were expressed as relative to the control levels with Tg treatment (E). *, P .05 relative to the control levels by Students t test.

To assess the roles of the PI3K and ERK-MAPK signaling cascades in the regulation of miR-16, miR-195, and miR-24 expression, FRTL-5 cells were treated with LY294002 or PD98059 in the presence of Tg, and then miRNA transcript levels were evaluated using real-time PI3K signaling regulates miRNAs and their target PCR analysis. As shown in Figure 4A, Tg suppressed the genes expression of all three miRNAs, as expected. PretreatIt has been reported that the Tg-induced growth of ment with PD98059 had no effect on the Tg-induced supFRTL-5 cells is suppressed by both the PI3K inhibitor pression. In contrast, LY294002 not only reversed the Tg LY294002 (10) and the MEK1 inhibitor, PD98059 (14). effect but also increased miR-16, miR-195, and miR-24 levels above A B C their untreated control values, sug Tg (+) miR-16 Mapk8 1.5 miR-195 gesting that PI3K-dependent, but 40 Ccne1 miR-24 Cdc6 not MEK-dependent pathways play 30 1.0 a role in the Tg reduction in miR-16, 20 miR-195, and miR-24. Effects of the Mapk8 0.5 10 PI3K and MAPK/ERK inhibitors on Ccne1 0 0 Mapk8, Ccne1, and Cdc6 were also Cdc6 assessed and compared with the exActb pression of a control gene, Actb (Figure 4B). Tg significantly increased the Tg (+) Tg (+) mRNA levels of the cell cycle-related Figure 4. PI3K is involved in Tg-mediated decreases of miRNAs and the increases of their target genes, especially those of Ccne1 and genes Ccne1 and Cd6. Quiescent FRTL-5 cells were treated with Tg in the presence of the MEK Cdc6, above the untreated control inhibitor PD98059 or the PI3K inhibitor LY294002 for 24 hours. A, miRNA levels were evaluated by real-time PCR. Expression of miRNAs was normalized to control samples without Tg treatment values, and PD98059 pretreatment and was expressed as the mean SEM (n 3). B and C, Expression of miRNA target genes was had no effect on the Tg-induced inevaluated by RT-PCR (B) and real-time PCR (C). The mRNA levels were normalized to control creases of these mRNA (Figure 4B) samples without Tg treatment and were expressed as the mean SEM (n 3). **, P .01 and protein levels (Figure 4C). relative to the control levels with Tg treatment by Students t test.
Relative miRNA levels Relative mRNA levels

3UTR of Myc. Overall, these findings suggest that the decrease of miR-24 expression by Tg could result in increased cell proliferation through cell cycle actions downstream of c-Myc induction.

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Relative mRNA levels 8 6 4 2 Control Control PD98059 LY294002 0

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Relative mRNA levels 40 30 20
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Figure 5. PI3K is involved in the Tg-mediated increase of target gene c-myc for miR-24. A and B, Quiescent FRTL-5 cells were treated with Tg in the presence of the MEK inhibitor PD98059 or PI3K inhibitor LY294002 for 24 hours. The mRNA expression of c-myc was evaluated by RT-PCR (A) and real-time PCR (B). The mRNA levels were normalized to control samples without Tg treatment and were expressed as the mean SEM (n 3). C, Quiescent FRTL-5 cells were treated with Tg for the indicated period of time. The mRNA expression of c-myc was evaluated by real-time PCR. The mRNA levels were normalized to control samples without Tg treatment and were expressed as the mean SEM (n 3). **, P .01 relative to the control levels with Tg treatment by Students t test.

LY294002, on the other hand, almost entirely abolished the Tg-induced elevations in Ccne1 and Cdc6 transcript (Figure 4, B and C, respectively). It did not, however, have an effect on the modest up-regulation of Mapk8 mRNA and protein by Tg (Figure 4, B and C, respectively). Consistent with the suppression miR-24 expression by PI3K but not MAPK/ERK activation, as shown in Figure 4A, the Tg-induced expression of its target gene Myc was significantly reduced (by about 50%) by LY294002, but not by PD98059 as demonstrated using both RT-PCR and real-time PCR analyses (Figure 5, A and B, respectively). The time-course of Myc induction following Tg showed that the increase in Myc transcript, although quite dramatic (40-fold increase over untreated control at 1h), is also transitory, with a return to control value after 24h (Figure 5C). This finding supports our contention that Myc escaped detection as a Tg-affected gene in the DNA microarray only because the dramatic Tg-induced increase in transcript level had returned to control value when the microarray assay was prepared.

Discussion
Tg stored in the follicular lumen is a potent suppressor of thyroid-specific genes and inductor of cell proliferation (27, 28). It has been shown through several lines of evidence that the effect of Tg is specific to 27S, 19S, and 12S Tg moieties and not due to some nonspecific effects of Tg or to substances(s) that coexist in the Tg preparations (10, 15, 16). However, the underlying molecular mecha-

nism(s), including the identification of the active regulatory element within Tg molecule, and the nature of the cellular Tg recognition system remain largely unknown (2, 5). In the present study, we investigated the involvement of specific miRNA species in the control of thyroid cell proliferation by Tg. We showed using a microarray containing 237 rat miRNAs followed by real-time PCR that Tg down-regulates the expression of 21 miRNAs to less than 50% of their original levels. Using the effect of miRNA overexpression on BrdU incorporation as a criterion, we identified only three miRNAs of the 21 (miR16, miR-195, and miR-24) as having an unambiguous role in the increase of thyroid cell proliferation in response to Tg. Of these three, miR-16 and miR-195 are also essential in conveying the TSH signal for increased proliferation of the thyroid cell, as we had demonstrated previously (27). In addition, three TSH-responsive genes (Mapk8, Ccne1, and Cdc6) that we had identified as targets of both miR-16 and miR-195 in the previous study were shown to be Tg responsive in the present report. Moreover, Tg-induced increases in the expression of these genes were significantly diminished by the overexpression of either miR-16 or miR-195, similar to the effect of their overexpression on TSH-induced increases of the same cell cycle-related genes noted in our earlier report (27). A third miRNA (miR-24) fulfilling the criteria for participation in Tg-induced cell proliferation had shown no involvement in TSH-stimulated proliferation in our previous report (27). This suggests that although TSH and Tg share at least two miRNAs in their control of thyroid cell proliferation, another miRNA, miR-24, may be limited to involvement in the proliferation pathways regulated by Tg. It is known that miR-24 suppresses Myc expression in other cell types, and we have shown this to be true in untreated FRTL-5 cells. We speculate that because Myc exerts control over progression through the cell cycle via its activation of specific cyclins and CDKs that Tg possesses an additional level of control over thyroid cell proliferation that is not shared with TSH. This could prove important in distinguishing autocrine signals (ie, Tg) for thyroid cell proliferation from endocrine signals, such as TSH, both of which use miR-16 and miR-195 to upregulate thyroid cell division and increase thyroid growth. The binding of TSHR results in the generation of cAMP. In turn, cAMP activates protein kinase A (PKA) to induce the expression of various genes necessary for cell proliferation. In our previous study, we showed that a specific inhibitor of PKA, H-89, partially reversed the TSH-induced decrease of miR-16 and miR-195 expression, indicating these two miRNAs are involved in the cAMP/PKA signal pathway of TSH-induced cell growth

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(27). Unlike TSH, Tg does not increase cAMP levels to increase cell replication, as evidenced by the fact that H-89 treatment does not alter the effect of Tg on cell growth (10). On the other hand, the specific PI3K inhibitor, LY294002, significantly reduces Tg-induced thyroid cell proliferation and also reverses both the TSH- and Tg-induced decreases in miR-16 and miR-195. These observations suggest that miR-16 and miR-195 are important to the up-regulation of cell proliferation by both TSH and Tg and that they act downstream of both cAMP and PI3K activation, by affecting the expressions of specific cell cycle-related genes. Although we showed that LY294002 could suppress the Tg-induced up-regulation of Ccne1, Cdc6, and Myc, we could not demonstrate an effect of the inhibitor on Mapk8 expression, suggesting that Mapk8 is regulated by other Tg-derived molecules or signal pathways that will require further examination. PI3K/Akt, however, is not the only signal pathway leading to a Tg-induced increase in the proliferation of thyroid cells. It was shown in a recent study (14) that Tg also activates the c-Raf/MEK/ERK arm of a MAPK-dependent pathway essential to increased cell proliferation. Specifically, it was shown that Tg increased the phosphorylation of c-RAF, MEK1/2, and ERK1/2 and furthermore that the MEK inhibitor, PD98059, strongly suppressed BrdU incorporation, indicating that this pathway, at least in part, mediates the Tg-induced increase in FRTL-5 cell proliferation. Because the transfer of phosphorylated ERK to the nucleus can activate transcription factors such as c-Myc, c-Fos, and c-Jun, the Raf/MEK/ERK pathway may have especial relevance to specific miRNAs that control the expression of these transcription factors. The existence of distinct PI3K and MAPK pathways transducing the effect of Tg on proliferation, only one of which (PI3K) is also activated downstream of TSH, is particularly intriguing in light of the three miRNAs that we found to be involved in Tg-induced thyroid cell proliferation, two of which (miR-16 and miR-195) also participate in TSH-induced proliferation. miRNAs act as negative regulators of gene expression by binding to the 3UTR region of their target mRNAs, with the lack of perfect complementarity allowing a single miRNA to target multiple genes. It has been approximated that one miRNA can potentially regulate more than 200 different genes, and many individual genes possess target sites for multiple miRNAs in their 3UTR, indicating the likelihood of a combinatory action of several miRNAs on gene activity (39). Our findings support the degenerate nature of miRNA regulation of cell function by demonstrating that miR-16 and miR-195 share the same target genes and act as common mediators for both TSH- and Tg-induced

cell growth, despite that fact that signal pathways used (cAMP and PI3K) do not completely overlap. miR-16 and miR-195 are members of miR-15 precursor family that also includes miR-15 and miR-497 (40). miR-24, the only miRNA regulated solely by Tg in the present study, is clustered with miR-23b and miR-27b on chromosome 17 in the rat and with those same two miRNA genes on chromosome 9 and 19 in the human (41). In humans, miR-15 and miR-16 are encoded on chromosome 13q14 (42) and have been shown to suppress expression of BCL2 (43). Deletions in this region have been reported to occur in about 50% of patients with B cell chronic lymphocytic leukemia (44). In the rat, the species of origin of our thyroid cell model, miR-16 and miR-195, are located on chromosomes 2 and 10, respectively, but as we report here, miR-16 appears to control cell division-related genes in FRTL-5 cells rather than proapoptotic genes. Bonci et al (45) have shown, on the other hand, that the miR-15a-miR-16 1 cluster targets both antiapoptotic (BCL2) and tumor promoter genes [CCND1 (encoding cyclin D1) and WNT3A] to control prostate cancer, suggesting that miR-16 1 is involved in the regulation of a wide range of actions that suppress growth. Of particular interest among these targets of miR-16 is cyclin D1. A recent study (46) showed that miR-195, closely related to miR-16 in the miR-15 precursor family, inhibits the proliferation of human glioma cells by directly targeting the 3UTR of both cyclin D1 and cyclin E1 genes. Cyclin E1 was identified as a target of both miR-16 and miR-195 in our present study. We surmise from these findings that direct targeting of either or both cyclins D1 and E1 by members of the miR-15 precursor family may represent a common mechanism for control of cell proliferation across species and cell phenotypes that is exploited by upstream signals from both TSH and Tg in the thyroid cell. At this point, we can only speculate as to how any of the three miRNAs identified with Tg-induced thyroid cell proliferation might be controlled by upstream activation of PI3K/Akt signaling. However, it is interesting to note that the tumor suppressor p53, which is inactivated downstream of PI3K/Akt activation (47), has also recently been shown to enhance the posttranscriptional maturation of miR16 1 (48). On the other hand, Fabbri et al (49) have demonstrated that the overexpression of miR16 1 in MEG-01 cells results in the decreased expression of p53 in what they suggest is a feedback circuit between the microRNA and the p53 transcription factor. It is possible then that Tg activation of PI3K in thyroid cells could activate cell-proliferation genes as a result of an Akt-induced degradation of p53, but proof of this

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supposition will require additional studies. Other transcription factors downstream of PI3K/Akt activation such as the forkhead transcription factor, Foxo3a (50), have been identified in the regulation of specific miRNAs, and these will also need to be taken into account. The demonstration of miR-24 involvement in Tg-, but not TSH-stimulated cell proliferation, suggests that specificity for one factor over another (eg, Tg vs TSH) in a particular aspect of thyroid cell function can be attained by the addition of a unique miRNA to the regulatory program. Thus, although both TSH and Tg can suppress miR-24 expression, only Tg induced- and not TSH-induced proliferation is reversed by overexpression of miR24, suggesting that action of this miRNA is restricted only to Tg-activated pathways of thyroid cell division. Furthermore, our study provides strong support for Myc, a gene that regulates cell growth, and whose expression we showed to be increased after Tg treatment (14), as a target of miR-24 suppression. A close association between miR-24 and both cell proliferation and carcinogenesis has been reported, although precise effects of the miRNA seem to depend on the specific cell type involved. For example, it has been reported that miR-24 directly regulates Myc and E2F2 to inhibit cell cycle progression in hematopoietic differentiation (38) and that it functions as a tumor suppressor in laryngeal squamous cell carcinoma (studied in vitro) through the down-regulation of S100A8 (51). Other investigators reported that miR-24 decreases p14ARF protein expression, thereby effectively blocking p53-mediated tumor suppression during the process of retinal tumorigenesis (52). It has also been reported that miR-24 down-regulates MAPK phosphatase-7 and enhances the phosphorylation of both JNK and p38 kinase to promote myeloid proliferation (53) and that it suppresses the expression of CDK inhibitor 1B (CDKN1B) via its modulation of dead end 1 (DND1) expression, leading to enhanced proliferation in tongue squamous cell carcinoma (54). A complete understanding of how miR-24 affects thyroid function and pathogenesis awaits further study, especially considering the apparently contrary reports that it is the most abundantly expressed miRNAs in the human healthy thyroid gland (55), but that it is also overexpressed, rather than underexpressed, in papillary thyroid carcinoma (56). In addition, other miRNAs, not identified in our present screening, may perform distinct roles in the regulation of normal or abnormal thyroid cell proliferation. One miRNA, miR-26a, reduced by Tg treatment in the present study but having no effect on Tg-induced proliferation in diploid FRTL-5 cells has been reported to decrease and regulate cell growth in anaplastic carcinoma (26). In addition, miR-221, another miRNA decreased by

Tg but having no effect on FRTL-5 cell proliferation, has been shown to be increased in papillary carcinoma and to promote cell proliferation (25) through the suppression of its target gene p27kip1 (57). Our present findings provide additional support for a functional role of miR-24 in the thyroid cell, specifically for a role in regulating TSH-independent proliferation, but whether this role also applies to abnormal cells is a subject for further investigation. In conclusion, we have shown here that Tg increases thyroid cell proliferation by suppressing the expression of specific miRNAs (miR-16 and miR-195), also used by TSH and perhaps other agonists, to increase thyroid cell division. Specifically, we have shown that both Tg and TSH activate PI3K signaling, decrease miR-16 and miR195 expression, and consequently up-regulate expression of the cell cycle-related genes, Mapk8, Ccne1, and Cdc6. On the other hand, the suppression of miR-24 expression is unique to Tg, as is the increased expression of its target gene Myc. Myc, in turn, would increase cell division through the activation of specific cyclins and other cell cycle-related proteins. These findings suggest that in addition to pathways of cell cycle regulation shared with TSH that Tg possesses unique means of regulating cell proliferation that are dependent on miR-24 and that could conceivably be involved in the autoregulation of thyroid function by Tg.

Acknowledgments
Address all correspondence and requests for reprints to: Koichi Suzuki, PhD, Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4 2-1 Aoba-cho, Higashimurayama-shi, Tokyo 189 0002, Japan. E-mail: koichis@nih.go.jp. This work was supported by part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant 33802400 to K.S.) . Disclosure Summary: The authors have nothing to disclose.

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