thyroid hormone regulation of mir-21 enhances migration ... · molecular and cellular pathobiology...

Molecular and Cellular Pathobiology

Thyroid Hormone Regulation of miR-21 Enhances Migrationand Invasion of Hepatoma

Ya-Hui Huang1, Yang-Hsiang Lin2, Hsiang-Cheng Chi2, Chen-Hsin Liao2, Chia-Jung Liao2,Sheng-Ming Wu2, Cheng-Yi Chen2, Yi-Hsin Tseng2, Chung-Ying Tsai2, Sheng-Yen Lin2,Yu-Ting Hung2, Chih-Jen Wang3, Crystal D. Lin4, and Kwang-Huei Lin2

AbstractThyroid hormone (T3) signaling through the thyroid hormone receptor (TRa1) regulates hepatoma cell growth

and pathophysiology, but the underlying mechanisms are unclear at present. Here, we have shown that theoncomir microRNA-21 (miR-21) is activated by T3 through a native T3 response element in the primary miR-21promoter. Overexpression of miR-21 promoted hepatoma cell migration and invasion, similar to that observedwith T3 stimulation in hepatoma cells. In addition, anti-miR-21–induced suppression of cell migration wasrescued by T3. The Rac-controlled regulator of invasion andmetastasis, T-cell lymphoma invasion andmetastasis1 (TIAM1), was identified as amiR-21 target additionally downregulated by T3. Attenuation and overexpression ofmiR-21 induced upregulation and downregulation of TIAM1, respectively. TIAM1 attenuation, in turn, enhancedmigration and invasion via the upregulation of b-catenin, vimentin, andmatrix metalloproteinase-2 in hepatomacells. Notably, correlations between TRa1, miR-21, and TIAM1 expression patterns in animal models paralleledthose observed in vitro. In the clinic, we observed a positive correlation (P¼ 0.005) between the tumor/nontumorratios of TRa1 and miR-21 expression, whereas a negative correlation (P ¼ 0.019) was seen between miR-21 andTIAM1 expression in patients with hepatoma. Our findings collectively indicate thatmiR-21 stimulation by T3 andsubsequent TIAM1 suppression promotes hepatoma cell migration and invasion. Cancer Res; 73(8); 2505–17.�2013 AACR.

IntroductionThe thyroid hormone (T3) is an important regulator of

growth, development, and differentiation in vertebrates. T3

binds to thyroid hormone receptors that belong to the nuclearreceptor superfamily. Human thyroid hormone receptors areencoded by TRa and TRb genes located on chromosomes 17and 3, respectively. The 2 genes yield several polypeptides viaalternative splicing and differential promoter usage. The TRa1,TRb1, andTRb2 genes encoding functional products have beenwell characterized. Accumulating evidence supports a criticalrole of thyroid hormone receptors in carcinogenesis. Forinstance, aberrant expression of thyroid hormone receptor

has been associated with human cancers (1). Earlier studiessuggest that partial loss of normal thyroid hormone receptorfunction occurs due to reduced expression or complete loss ofactivity resulting in mutations and/or aberrant expression,which in turn, provides an opportunity for tumor cells toproliferate, invade, and metastasize (2).

MicroRNAs (miRNA) are small approximately 22-nucleotideRNA molecules that modulate gene expression via the RNAinterference (RNAi) pathway. MiRNAs generally exhibit partialcomplementarity, and often bind within the 30 untranslatedregion (30UTR) of target mRNAs, leading to translationalrepression and/or degradation (3). Inappropriate expressionof miRNAs is strongly associated with carcinogenesis, as thegenomic aberrations observed in cancers directly reflectmiRNA expression patterns. Moreover, increasing evidenceshows that miRNA gene expression is dysregulated in humancancers (4, 5). Specific over- or underexpression of miRNA iscorrelated with particular tumor types (6, 7). To date, a numberof miRNAs that seem to function as tumor suppressors oroncogenes have been identified.

A number of earlier studies have reported that T3/thyroidhormone receptor signaling plays a critical role in hepatoma(8–10). In addition, miRNAs play a fundamental role in regu-lating gene expression in multicellular eukaryotes. Therefore,to clarify the functions of specific transcription factors, miR-NAs can be evaluated as potential targets. In hepatoma,miRNAexpression is frequently dysregulated, and specific miRNAshave been shown to regulate migration and invasion (11). It is

Authors' Affiliations: 1Liver Research Center, Department of Hepato-Gastroenterology, ChangGungMemorial Hospital, Linkou; 2Department ofBiochemistry, College of Medicine, Chang-Gung University, Taoyuan;3Department of Neurosurgery, Kaohsiung Medical University Hospital,Kaohsiung Medical University, Kaohsiung, Taiwan; and 4Pre-med Pro-gram, Pacific Union College, Angwin, California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Y.H. Huang and Y.H. Lin contributed equally to this work.

Corresponding Author: Kwang-Huei Lin, Department of Biochemistry,College of Medicine, Chang-Gung University, 259 Wen-hwa 1 Road,Taoyuan, Taiwan. Phone: 886-3-2118263; Fax: 886-3-2118263; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-2218

�2013 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 2505

on October 6, 2020. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 26, 2013; DOI: 10.1158/0008-5472.CAN-12-2218

http://cancerres.aacrjournals.org/

speculated that miRNAs are critical downstream targets ofthyroid hormone receptors that participate in hepatoma cellprogression. To identify the specific miRNA targets of thyroidhormone receptor, 270 miRNA expression profiles in HepG2–TRa1 cells were obtained in the presence or absence of T3

using stem-loop quantitative reverse transcription PCR(qRT-PCR) analysis. Consequently, several miRNAs up- ordownregulated upon T3 stimulation were detected. We ulti-mately focused on miR-21, one of the T3-modulated miRNAsthat is dysregulated in various cancers. Direct regulation ofmiR-21 by T3 was confirmed, and the mechanism underlyingthe effects of T3 on hepatoma cell migration furtherinvestigated.

Materials and MethodsqRT-PCR for miRNA and mRNA

The qRT-PCR method for detection of mature miRNAs andmRNAs was conducted as described previously (10, 12).

Cell culture and transfectionThe human hepatoma cell lines, HepG2, Hep3B, SK-Hep1

(obtained fromAmerican Type Culture Collection), and J7 (giftfrom Dr. C.S Yang, National Taiwan University, Taiwan; ref. 13)were routinely grown in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% (v/v) FBS. The cell lines haverecently been authenticated using the StemElite ID System(Promega). TRa1- and TRb1–overexpressing cell lines werecultured, as described previously (14). The serumwas depletedof T3 (Td) using an earlier procedure (15). Cellswere cultured at37�C in a humidified atmosphere of 95% air and 5% CO2.

Patients and statistical methodsTo determine the associations among TRa1, miR-21, and

TIAM1 in hepatoma, total RNAs of tumor and adjacent non-tumor liver tissues from 40 patients with hepatoma (13 femalesand 27 males) from the Taiwan Liver Cancer Network wereobtained for analysis. The basic clinical characterization of the40 patients is presented in Supplementary Table S1. The studyprotocol was approved by the Medical Ethics and HumanClinical Trial Committee of the Chang Gung Memorial Hos-pital (IRB No. 98-0798B).

Stable transfection of Hep3B cells with TIAM1knockdown

RNAi against TIAM1 (obtained from Academia Sinica inTaiwan), pLKO.1-shTIAM1, was used to deplete endogenousTIAM1 in Hep3B cells. Specifically, the Hep3B cells weretransfected with pLKO.1-shTIAM1 using Turbofect (Fermen-tas). Transfected cells were incubated and selected in DMEMsupplemented with 10% FBS containing 0.3 mg/mL puromycinfor at least 2 weeks until colonies could be picked.

Amplified chromatin immunoprecipitation assayThe chromatin immunoprecipitation (ChIP) assay was con-

ducted as described in a previous report (8). The upstream T3

response element (TRE) region of the pri-miR-21 gene wasamplified with the forward primer, 50-AGAGGGCGGG-CAGTTTCTT-30, starting at nucleotide �1017, and reverse

primer, 50-AATAAGGAAATGACTTATGC-30, starting at nucle-otide �958.

Immunoblot analysisProteins were loaded on a 6% or 10% SDS-polyacrylamide

gel for electrophoresis before transfer to a polyvinylidenedifluoride membrane (PerkinElmer). The membrane wasblotted with antibodies specific for thyroid hormone receptorprotein (C4; ref. 16), TIAM1 (Calbiochem), vimentin, b-cate-nin, phospho-b-catenin (Santa Cruz), c-myc (GeneTex), cyclinD1, c-Jun (Epitomics), b-actin, and glyceraldehyde 3-phos-phate dehydrogenase (GAPDH; Chemicon) for detection.Blots were incubated with horseradish peroxidase-conjugatedsecondary antibody, and developed using an ECL detectionkit (Millipore Corp.). Immunoblot assays were conductedindependently at least 3 times. The intensities of the immu-noreactive bands were quantified using Image Gauge software(Fuji Film).

30UTR luciferase reporter assayFull-length TIAM1 (1950 nt) or MSH2 (272 nt) 30UTR (Sup-

plementary Fig. S1) was synthesized and cloned into the pMIR-REPORT vector (Applied Biosystems) containing the luciferasereporter gene (TIAM1 orMSH2 30UTRwt). The putativemiR-21recognition sites in TIAM1 or MSH2 30UTR were subjected tosite-directed mutagenesis (TIAM1 or MSH2 30UTR mut), andthe mutated sequences were validated via automated DNAsequencing. The pCDH-CMV-MCS-EF1-copGFP and pmiRZiplentivector (SBI System Biosciences) was used to express pre-miR-21 and antisense miR-21 small RNA, respectively. Todetermine the effects of miR-21 on TIAM1 30UTR, pMIR-TIAM1-30UTR and pre-miR-21, anti-miR-21 or negative controlvector were cotransfected into HepG2 cells using TurboFect.Similarly, HepG2 cells cotransfected with pMIR-MSH2-30UTRand pre-miR-21 or negative control vector were examined. Todetermine the effects of T3 on TIAM1 30UTR, HepG2–TRa1cells precultured in Td medium were transfected with pMIR-TIAM1-30UTR (wt) in the presence or absence of T3. Aftertransfection for 24 hours, cells were extracted for detection ofluciferase activity.

Migration and invasion assayCells (5 � 104/200 mL) in serum-free DMEM were seeded

onto the top chambers of Transwell filter devices and DMEMcontaining 10% FBS added to the bottom chambers, asdescribed previously (14).

Gelatin zymographyAn aliquot of concentrated medium (20 mg) from Hep3B–

shNeo and Hep3B–shTIAM1 cell cultures was loaded onto a10% nondenaturing polyacrylamide gel containing 0.1% gela-tin, as described previously (17).

Animal modelInModel I, male Sprague Dawley (SD) rats were subjected to

thyroidectomy (Tx) at 6 weeks of age according to a previousmethod, with a view to examining the association between T3

/thyroid hormone receptor and miR-21 in vivo (10). To

Huang et al.

Cancer Res; 73(8) April 15, 2013 Cancer Research2506




investigate the correlation between T3/thyroid hormonereceptor and miR-21 in T3-enhanced metastasis of hepatomacells in vivo, J7-TRa1 xenografts of eu-, hypo-, and hyperthyroidsevere combined immunodeficiency (SCID) mice generated ina previous study by our group (18, 19) were used (Model II).Finally, to determinewhether the negative correlation betweenthe miR-21 and TIAM1 can be found in vivo, knockdown ofmiR-21 in SK-Hep1 cells was injected into SCIDmice via the tailvein (Model III). All animal experiments were conducted inaccordance with the National Institutes of Health Guide andChang-Gung Institutional Animal Care and Use CommitteeGuide for the Care and Use of Laboratory Animals (IACUCApproval No. CGU09-011).

Statistical analysisPhi correlation coefficients were computed to determine the

correlations among the tumor/nontumor (T/N) ratios of TRa1,miR-21, and TIAM1. Statistical analysis was conducted usingSPSS version 15.0. Other statistical analysis of data was con-ducted using Student t test. Data were considered statisticallysignificant at P less than 0.05. Values are presented asmeans�SD of at least 3 independent observations.

ResultsExpression profiles ofmiRNA inHepG2 cells treatedwithor without T3

Because of low expression of endogenous TRa1 in varioushepatoma cell lines (Supplementary Fig. S2), a HepG2 cell lineexpressing high levels of TRa1 (HepG2–TRa1) was establishedto identify the miRNA genes potentially regulated by T3. Intotal, 14 miRNAs potentially regulated by T3 were identified.Ten of these miRNAs were upregulated by at least 3-fold, and 4miRNAswere downregulated by at least 2-fold (SupplementaryTable S2). Among the 14 miRNA candidates, miR-21 has beenshown to be upregulated in various cancers, including hepa-toma (7), suggestive of a role as a central oncomir (20). A pilotstudy additionally revealed miR-21 upregulation by T3 inHepG2–TRa1 cells. Interestingly, T3/thyroid hormone recep-tor signaling may exert both oncogenic (10) and tumor sup-pressor effects (21, 22). The findings thatmiR-21 is increased inhepatoma tissue and potentially upregulated as a target of T3 inHepG2 cells imply that T3 functions as an oncogene in this case.

Validation of T3-mediated upregulation of miR-21 inHepG2–TR cellsTo confirm the positive regulation of miR-21 by T3, HepG2

cells stably expressing TRa1 (HepG2–TRa1) and TRb1(HepG2–TRb1) were examined. A HepG2 cell line expressingthe empty vector, Neo, was used as the control (HepG2–Neo).thyroid hormone receptor expression levels of the 3 stableHepG2 cell lines are shown in Fig. 1A. The observed increase inmiR-21 expression after T3 treatment in the HepG2–TR celllines was both time- and dose-dependent (Figs 1B and C). Incontrast, no changes inmiR-21 levels were evident following T3

treatment in the HepG2–Neo cell line (Fig. 1D). Moreover,expression of themiR-21 primary transcript (pri-miR-21, acces-sion No. AY699265, Supplementary Fig. S3) was stimulated ina time- and dose-dependent manner in HepG2–TRa1 and

HepG2–TRb1 cells treated with T3 (Fig. 1E and SupplementaryFig. S4). Accordingly, we speculated that the promoter region ofmiR-21 possesses a TRE. ThemiR-21 gene is located within theintron of TMEM49. The TMEM49 level in T3-treated HepG2–TRa1 cells was thus examined to eliminate the possibility thatincreased miR-21 expression is attributed to T3-regulatedTMEM49. As shown in Fig. 1F, TMEM49mRNA expression wassignificantly increased by 1.5- and 1.7-fold in HepG2–TRa1cells treatedwith 10 nmol/LT3 for 48 and 72 hours, whereas themiR-21 level was elevated approximately 4.3- and 5.9-fold underthe same conditions, respectively (Fig. 1B). This difference inrelative fold change between miR-21 and TMEM49 levels inHepG2–TRa1 cells (4.3-fold vs. 1.5-fold and 5.9-fold vs. 1.7-foldfollowing 10 nmol/L T3 treatment for 48 or 72 hours, respec-tively) was significant. The differences in fold change ofmiR-21(both primary andmature forms) andTMEM49 induction byT3

are indicative of direct regulation of miR-21 expression by T3,rather than coregulation with TMEM49.

Characterization of TRE in the miR-21 geneTo determine whether the promoter region of pri-miR-21

contains TRE, we analyzed the 3,000 nucleotides regionupstream of the gene using Vector NTI software. Seven puta-tive TREs were identified in the pri-miR-21 promoter region(�3000/þ1; Supplementary Fig. S5). Among these, 2 putativeregions (TREA andB) were predicted to possess palindromic(Pal) TRE, 3 (TREC, F andG) to include inverted palindrome(Lap) TREs, and TREE to contain direct repeat 4 (DR4) TRE. Inaddition, 2 overlapping response elements of TRED (Lap andPal) were predicted (Fig. 2A and B). A luciferase-based reporterplasmid containing the promoter region (�3000/�45) wasconstructed for the luciferase promoter assay. An approximate3- to 5-fold increase in luciferase activity was induced by T3 (10and 100 nmol/L) in HepG2–TRa1 cells (Fig. 2A, I), indicating aresponse of the promoter region to T3/thyroid hormone recep-tor. To further identify the putative TREwithin the�3,000/�45region that responds to T3/thyroid hormone receptor, serialfragments (II to VII) were constructed for an additionalpromoter assay. Among these fragments, only 2 containing theTRED region (fragments III and V) displayed the luciferaseactivity under T3 treatment conditions. In addition, the lucif-erase activity was dependent on the T3 dose (Fig. 2A). Ourluciferase promoter assay results indicate that the �1,109/�875 region of the pri-miR-21 promoter possesses native TRE.TheChIP assaywas subsequently conducted to further confirmthe association of the �1,109/�875 region with T3/TR in vivo.The thyroid hormone receptor and RXR binding region wasnarrowed down to positions�1,017/�958 within this region ofthe pri-miR-21 promoter (Fig. 2C, right). The native TREof furinassociated with thyroid hormone receptor and RXR was usedas a positive control (Fig. 2C, left), whereas additive IgG wasused as a negative control in the ChIP assay. In summary, ouranalytical results show thatmiR-21 is a direct thyroid hormonereceptor target gene–containing native TRE.

Prediction of miR-21 targetsThe possible effects andmechanisms of action of T3/thyroid

hormone receptor-induced miR-21 in hepatoma cells were

Regulation of miR-21 by Thyroid Hormone Receptor

www.aacrjournals.org Cancer Res; 73(8) April 15, 2013 2507




further analyzed. Specific potential miR-21 target genes neg-atively regulated by T3 were selected as candidates for study. Intotal, 307 genes were predicted as miR-21 targets using Tar-getScan software (http://www.targetscan.org/). Followingcomparison with 3,029 genes potentially downregulated byT3 in HepG2–TRa1 cells analyzed previously with cDNAmicroarray (data not shown), 62 were selected. Among these,5 genes (cyclin-dependent kinase: CDK6, mismatch repairprotein: MSH2, programmed cell death 4: Pdcd4, TGF-b typeII receptor: TGFBR2, and T-cell lymphoma invasion andmetastasis 1: TIAM1) were confirmed as miR-21 targets (23–27). Moreover, in view of the enhanced capability of hepatomacell migration after T3 application (14), Pdcd4 and TIAM1 arereported to be associated with cell migration and invasion.Earlier research has shown that miR-21 promotes migrationand invasion through a miR-21-PDCD4-AP-1 feedback loop inhepatoma cells (28). Furthermore, a paradoxical role of TIAM1in migration of various cancers has been documented (23, 29).Accordingly, we focused on TIAM1 for further study to validate

its role in liver carcinogenesis. The issue of whether TIAM1 isalso a direct target of miR-21 in hepatoma cells remains to beestablished. Two sequences with 7 nucleotides (AUAAGCU) ofTIAM1 30UTR, identified as the seed region of miR-21, werepredicted using TargetScan. Full-length TIAM1 30UTR contain-ing 2 seed regions located at positionsþ202/þ208 andþ1861/þ1867 were cloned into a cytomegalovirus (CMV)-drivenluciferase reporter plasmid. In the presence of TIAM1 30UTR,miR-21 suppressed luciferase activity. This suppressive effectwas diminished uponmutation of the 2miR-21 binding sites inHepG2 cells (Fig. 3A, left). In addition, knockdown of endog-enous miR-21 (anti-miR-21) in the presence of TIAM1 30UTRinduced luciferase activity, which was also decreased uponmutation of the seed region of TIAM1 30UTR (Fig. 3A, right). Tovalidate the effects of TIAM1 protein expression in the pres-ence of miR-21 in hepatoma cells, Hep3B cells expressinghigher levels of TIAM1 than HepG2 cells were employed forfurther analysis (Fig. 3B, lanes 1 and 2).We observed a decreasein TIAM1 protein expression in the presence ofmiR-21 (Fig. 3B,

Figure 1. Upregulation of miR-21by T3 in HepG2–TRs cells. A,expression of the thyroid hormonereceptor (TR) protein in HepG2–Neo, HepG2–TRb1, and HepG2–TRa1 cell lines. B–F, cells wereincubated for 24, 48, and 72 hoursin the absence or presence of T3(1, 10 nmol/L). Expression ofmature miR-21 in HepG2–TRa1(B), HepG2–TRb1 (C), and HepG2–Neo (D) cells using stem-loopqRT-PCR. E, expression of primarymiR-21 in HepG2–TRa1 cells in thepresence or absence of T3. F,TMEM49 mRNA expression inHepG2–TRa1 cells in the presenceor absence of T3. Values are mean� SD from 3 independentexperiments, each conducted induplicate. �, P < 0.05; ��, P < 0.01.

Huang et al.





lanes 4 vs. 3), and conversely, increased TIAM1 expressionupon knockdown of endogenousmiR-21 (using anti-miR-21) inHep3B cells (Fig. 3B, lanes 6 vs. 5). On the basis of thesefindings,we propose that TIAM1 is a direct miR-21 target in hepatomacells.

Downregulation of TIAM1 by T3/thyroid hormonereceptor in Hep3B cellsAsmiR-21 is upregulated by T3, we further examined wheth-

er TIAM1 30UTR luciferase activity is affected by the T3. Asshown in Fig. 3C, a significant decrease in TIAM1 30UTRluciferase activity was observed in HepG2–TRa1 cells in thepresence of T3, compared with that in the absence of T3.Subsequently, Hep3B cells expressing high levels of endoge-nous TIAM1 were used to overexpress TRa1 (Hep3B–TRa1, Fig. 3D) for the purpose of validating TIAM1 regulationby T3. The dose-dependent upregulation of miR-21 in T3-trea-ted Hep3B–TRa1 (Fig. 3E) was similar to that in HepG2–TRcells (Figs 1B and C). T3-mediated repression of the TIAM1protein level was observed in Hep3B–TRa1 cells (Fig. 3F, lanes1–4). To verify that the effect of T3 on TIAM1 is mediated, atleast in part, through miR-21, knockdown of endogenous miR-21 (using anti-miR-21) in Hep3B–TRa1 cells was conducted.Notably, suppression of TIAM1 protein in Hep3B–TRa1 cells

by T3 was rescued in cells depleted of endogenousmiR-21 (Fig.3F, lanes 6 vs. 8). The data presented in Fig. 3 suggest that thenegative regulation of TIAM1 by T3 occurs through the T3

-mediated upregulation of miR-21 in hepatoma cells. MSH2, atumor suppressor downregulated by T3/thyroid hormonereceptor signaling and miR-21, was additionally examined. Asshown in Supplementary Fig. S6A, the luciferase activity of full-length MSH2 30UTR (272 nt) containing a seed site (positionsþ33/þ39) was inhibited by miR-21, which was partiallyrestored upon mutation of the seed region. Moreover, MSH2expression was suppressed in the presence of T3 or uponoverexpression of miR-21 (Supplementary Fig. S6B and C,lane 2), and conversely, increased upon knockdown of miR-21(anti-miR-21, Supplementary Fig. S6C, lane 4).

Correlations among TR,miR-21, and TIAM1 in hepatomapatients

To determine whether thyroid hormone receptor, miR-21,and TIAM1 expression patterns are correlated in hepatoma,total RNA from paired tumor and adjacent nontumor livertissues was extracted from a series of 40 consecutivepatients with hepatoma. The T/N ratios of TRa1, miR-21,and TIAM1, assessed using qRT-PCR, are presented in Sup-plementary Table S3. TRa1 RNA expression was higher in

Figure 2. The promoter region of miR-21 contains native TRE as a direct T3/TR target gene. A, HepG2–TRa1 cells were transfected with luciferase reportervectors with (pA3TK-Luc) or without (pGL3-Basic) aminimal thymidine kinase promoter expressing full-length and various fragments of pri-miR-21 promoter.Luciferase activity was examined after cells were incubated for 24 hours in the presence or absence of T3 (10, 100 nmol/L). The vector expressingb-galactosidase was used to normalize transfection efficiency control. Values are mean � SD from 3 independent experiments, each conducted intriplicate. �� P < 0.01. B, the type of putative TREs in the pri-miR-21 promoter region presented in A. C, binding of TRa1, together with RXR to thepri-miR-21 (right) and furin (positive control, left) promoter. Chromatin fragments were prepared from HepG2–TRa1 cells. The ChIP assay wasconducted using control IgG, anti-TR, or anti-RXR antibody. Chromatin fragments without IgG, anti-TR, or anti-RXR antibody were used as an inputcontrol. Two sets of primers for positive control TRE (furin) and pri-miR-21 TRE were used for PCR and gel electrophoresis.






87.5% (35/40) of tumor compared with nontumor tissues.In 35 patients with hepatoma with increased TRa1 expres-sion in tumor tissues, miR-21 was concomitantly increasedin 80.0% (28/35) of the tumor regions. In addition, TIAM1RNA expression was decreased in 71.4% (20/28) of tumortissues in 28 patients with hepatoma displaying the upre-gulation of TRa1 and miR-21. Phi correlation coefficientsrevealed a significant positive correlation between the T/Nratios of TRa1 andmiR-21 (w¼ 0.444, P¼ 0.005). Moreover, asignificant negative correlation between the T/N ratios ofmiR-21 and TIAM1 was observed (w ¼ �0.370, P ¼ 0.019).Our analytic results imply that the correlations among TRa1,miR-21, and TIAM1 expression patterns in patients withhepatoma are similar to those observed in hepatoma cellsin vitro.

T3 and miR-21 enhance Hep3B cell migrationStimulation of T3 promotes migration in HepG2 cells (14).

Consistent with this finding, an earlier study has shownthat a miR-21 inhibitor suppresses migration of Huh7 cells(30). To establish the migration properties of Hep3B cellstreated with T3 or displaying elevatedmiR-21 expression, the

Transwell assay was conducted. Our data showed thatmigration of Hep3B–TRa1 cells stimulated with T3 isenhanced (Fig. 4Aa). Similarly, cell migration (Fig. 4Ab) andinvasion (Fig. 4B) were promoted in miR-21-overexpressingHep3B cells. Conversely, cell migration was inhibited inHep3B cells depleted of endogenous miR-21 (Fig. 4Ac).Epithelial-mesenchymal transition (EMT) has been pro-posed as an index of enhanced metastasis in various malig-nant tumor types (31). It is suggested that the EMT markers,vimentin and b-catenin, play important roles in metastasisof hepatoma (32, 33). Interestingly, increased expression ofthese 2 molecules was observed in not only Hep3B–TRa1cells treated with T3, but also in miR-21-overexpressingHep3B cells (Fig. 4C, lanes 1–5). The downstream targetproteins of b-catenin, c-myc, and cyclin D1, were addition-ally elevated by T3 (Fig. 4C). b-Catenin was significantlyupregulated (by 1.8-fold) in miR-21-overexpressing Hep3Bcells, compared with the control cell line (Fig. 4C lanes 5 vs.4), and significantly downregulated (by 60%) in the miR-21knockdown cell line (Fig. 4C, lanes 7 vs. 6). Moreover,vimentin was downregulated upon knockdown of endoge-nous miR-21 (Fig. 4C, lanes 7 vs. 6). Our results collectively

Figure3. TIAM1 is amiR-21 target downregulated byT3. A, relative luciferase activity levels inHepG2cells cotransfectedwithmiR-21 (left) or anti-miR-21 (right)and TIAM1wild-type (wt) or mutant-type (mut) 30UTR vector. The luciferase activity was normalized to that of b-galactosidase. Data are presented as relativefold change to pCDH or pmiRZip control vector-transfected cells (Scrambled). B, endogenous TIAM1 protein levels in HepG2 and Hep3B cells (left).Endogenous TIAM1 protein levels in Hep3B cells expressing miR-21 (middle) or anti-miR-21 (right). Cells expressing pCDH (middle) or pmiRZip (right)as an internal control (Scrambled). C, relative luciferase activity levels in HepG2–TRa1 cells transfected with TIAM1 30UTR vector were incubated for 24 hoursin the absence or presence of T3 (100 nmol/L). D, expression of the TRa1 protein in Hep3B–Neo and Hep3B–TRa1 cell lines using immunoblotassay. E, expression of mature miR-21 in Hep3B–TRa1 cells incubated for 24 hours in the absence or presence of T3 (10, 100 nmol/L) using stem-loopqRT-PCR. F, endogenous TIAM1 protein levels in Hep3B–TRa1 cells incubated for 48 and 72 hours in the absence or presence of T3 (10 nmol/L; left).The protein expression of TIAM1 in Hep3B–TRa1 cells with or without miR-21 knockdown incubated for 48 hours in the absence or presence ofT3 (10 nmol/L; right). Values are mean � SD from 3 independent experiments, each conducted in duplicate. �, P < 0.05; ��, P < 0.01.

Huang et al.





Figure 4. MiR-21 enhances themigration and invasion of Hep3Bcells. A, Hep3B–TRa1 cell migrationability in the absence (Td) orpresence of T3 (T3; a). Hep3B cellswere transfected with miR-21 (b) oranti-miR-21 (c) for 72 hours. Hep3Bcells transfected with pCDH orpmiRZip vector (scrambled) wereused as controls. The migrationability of transfected cells wasdetermined using the Transwellmigration assay. B, invasive ability ofHep3B cells transfected withmiR-21or control vector (pCDH) using thematrigel-coated Transwell assay.Quantification of migratory orinvasive cells is presented on theright. Data are from 3 independentexperiments, each conducted induplicate. �, P < 0.05; ��, P < 0.01.C, expression of vimentin, b-catenin,c-myc, cyclin D1, and TRa1 inHep3B–TRa1 cells in the absence orpresence of T3 (left). Expression ofvimentin and b-catenin in Hep3Btransfected with miR-21 (middle) oranti-miR-21 (right). GAPDH is aninternal control.






indicate that the effects of miR-21 and T3 on cell migrationand metastasis-associated molecules (such as vimentin andb-catenin) are similar in Hep3B cells.

TIAM1 knockdown in Hep3B cells promotes migrationand invasion

To investigate the effects of TIAM1 on cell migration and theunderlying signaling pathways, we conducted knockdown ofTIAM1 in Hep3B or SK-Hep1 cells. The TIAM1 protein levels in4 Hep3B stable cell lines are shown in Fig. 5A. The b-catenin

level was significantly enhanced (by 1.6- to 1.7-fold) in the 2TIAM1 knockdown Hep3B cell lines (shTIAM1; K1, K2), com-pared with the control cell line (shNeo; N1, N2; Fig. 5A). Thephosphorylated b-catenin level was downregulated by almost40% in the K1 andK2 cell lines. Immunoblot analysis revealed amarked increase in the expression levels of the b-catenin targetproteins (c-myc, cyclin D1, and c-Jun) in the 2 cell lines(Fig. 5A). In addition, vimentin expression was increased inHep3B cells depleted of TIAM1 (Fig. 5A). K1 and K2 cell linesdisplayed enhanced activity (by 1.4- to 3.6-fold) of pro-matrix

Figure 5. Knockdown of TIAM1 promotes migration and invasion of Hep3B cells. A, expression of TIAM1, vimentin, b-catenin, phospho-b-catenin, c-myc,cyclin D1, and c-Jun protein in Hep3B–shNeo#1 (N1), Hep3B–shNeo#2 (N2), Hep3B–shTIAM1#1 (K1), and Hep3B–shTIAM1#2 (K2) cell lines. B,expression of proMMP2 inHep3B–shNeoandHep3B–shTIAM1cell lines using zymographassay.C,migration abilities ofHep3B–N1,Hep3B–N2,Hep3B–K1,and Hep3B–K2 cells assessed using the Transwell migration assay. D, quantification of migratory cells fromC. E, invasion abilities of Hep3B–N1, Hep3B–N2,Hep3B–K1, and Hep3B–K2 cells using the matrigel-coated Transwell assay. F, quantification of invasive cells from E. Values are mean � SDfrom 3 independent experiments, each conducted in duplicate. �, P < 0.05; ��, P < 0.01.

Huang et al.





metalloproteinase (MMP) 2 (72 kDa; Fig. 5B). Moreover, migra-tion (Figs 5C and D) and invasion (Figs 5E and F) abilities wereincreased approximately 3.1- to 6.0-fold and 1.5- to 2.8-fold,respectively, in these cell lines. To further verify the effect ofmiR-21 on metastasis in vivo, SK-Hep1 cells possessing highmetastatic ability and endogenous TIAM1 expression wereused. Expression ofmiR-21 was stimulated by T3 (Supplemen-tary Fig. S7A), whereas TIAM1 and MSH2 levels weredecreased.Moreover, c-myc and cyclin D1were upregulated byT3 (Supplementary Fig. S7B), and b-catenin was significantlydownregulated in anti-miR-21–expressing cells (Supplemen-tary Fig. S7C). Phosphorylated b-catenin protein was upregu-lated in the anti-miR-21–expressing cell line. Expression levelsof c-myc, cyclin D1, and vimentin were significantly decreased(Supplementary Fig. S7C). Our data collectively indicate thatmiR-21 stimulation by T3 and subsequent TIAM1 suppressionpromotes cell migration and invasion in hepatoma cells,possibly through the upregulation of b-catenin, c-myc, c-jun,and vimentin (Fig. 5 and Supplementary Fig. S7).

Suppression of cell migration induced by miR-21knockdown is rescued by T3 in Hep3B–TRa1 cellsTo confirm the finding that T3/thyroid hormone receptor

signaling promotes cell migration via T3-mediated positiveregulation of miR-21, Hep3B–TRa1 cells depleted of miR-21were examined in the presence or absence of T3 using theTranswell migration assay. As shown in Fig. 6, the cell migra-tion was markedly promoted in the presence of T3, comparedwith that in the absence of T3 (Figs 6A, I vs. II and 6B). The cellmigration effect in the absence of T3 was significantly sup-pressed after miR-21 knockdown (Fig. 6A, I vs. III and 6B),which was rescued following administration of T3 (Figs 6A, IIIvs. IV and 6B).Moreover, promotion of cell migration by T3wasmarkedly repressed after miR-21 knockdown (Fig. 6A, II vs. IVand 6B). These results show that T3 rescues anti-miR-21–mediated suppression of cell migration. Furthermore, T3

-induced cell migration is inhibited by anti-miR-21. On thebasis of these findings, we propose that T3-mediated positiveregulation of miR-21 promotes Hep3B cell migration.

Correlations among TR, miR-21, and TIAM1 in vivoTo investigate whether the effects of miR-21 in vitro can be

replicated in vivo, 3 animal models were designed. First, sur-gical thyroidectomy was conducted in 2 groups of 6-week-oldmale SD rats to determine the in vivo responses of miR-21 toT3 treatment.One group (TxþT3)was injecteddailywithT3 for2 weeks, the second group (Tx) received no T3 injections, andthe third group included sham-operated controls. Rats weresacrificed at the end of the experiment (4 weeks after thyroid-ectomy), and serum was collected for T3 and thyroid-stimu-lating hormone determination, as described previously (21).Stem-loop qRT-PCR analysis disclosed that miR-21 expressionin the TxþT3 group was 2.8-fold compared with the shamgroup, and conversely, 0.7-fold compared with sham group inthe Tx group (Fig. 7A), indicative of a positive correlationbetween T3/thyroid hormone receptor and miR-21 in vivo.Second, to determine whether the in vitro metastatic effect

of miR-21/T3 occurs in vivo, SCID mice were injected with J7-

TRa1 (a highly metastatic hepatoma cell line). Animals weresubjected to hyperthyroid, euthyroid, and hypothyroid condi-tions after injection (18, 19). The hyperthyroid group of SCIDmice injected with J7-TRa1 cells displayed higher miR-21expression (Fig. 7B), but lower TIAM1 expression comparedwith euthyroid (control) group (Fig. 7C, IX vs. III). Conversely,the hypothyroid group displayed decreased miR-21 (Fig. 7B)and higher TIAM1 expression (Fig. 7C, VI vs. III). Tumors aremarked with black lines. Figure 7C, I, II, IV, V, VII, and VIIIrepresent hematoxylin and eosin (H&E) staining, with II, V,and VIII depicting a higher magnification than I, IV, and VII,respectively. Finally, SCID mice with miR-21–depleted SK-Hep1 (anti-miR-21) xenografts developed fewer metastaticfoci in lungs, as evident from H&E staining (Fig. 7D, III), witha 98.2% decrease in metastatic index (tumor density per cm2;Fig. 7E), compared with SK-Hep1–scrambled xenografts(Fig. 7D, I, and E). Moreover, a stronger dark brown colorrepresenting TIAM1 protein was observed in the tumor areasof SK-Hep1-anti-miR-21 xenografts (Fig. 7D, IV, arrowhead),compared with SK-Hep1–scrambled xenografts (Fig. 7D, II).

Figure6. Anti-miR-21-repressed cellmigration is rescuedbyT3 inHep3B–TRa1 cells. A, Hep3B–TRa1 cells were transfected with the pmiRZip(scrambled; I and II) or pmiRZip-21 (anti-miR-21; III and IV) vector, andincubated for 24 hours in the absence (I and III) or presence (II and IV) of T3for the Transwellmigration assay. B, quantification ofmigratory cells fromA. Values are mean � SD from 3 independent experiments, eachconducted in duplicate. ��, P < 0.01.






Analogous results were obtained with both in vivo animal andcell culture models.

DiscussionT3/thyroid hormone receptor signaling promotes cell inva-

siveness involved in hepatoma progression through the director indirect upregulation of several genes, including furin (10),methionine adenosyltransferase 1A (9), plasminogen activator

inhibitor-1 (18), and TNF-related apoptosis-inducing ligand(19). MiRNAs are small RNAmolecules processed from endog-enous precursor RNA with stem-loop structures that negative-ly regulate gene expression. To date, several tumor progres-sion-related genes have been identified as specific miRNAtargets (34, 35). Aberrant miRNA expression has been reportedin various tumors, including liver cancer (36, 37). Data from thecurrent study indicate that both T3/thyroid hormone receptorsignaling and miRNAs affect tumor cell progression through

Figure 7. Correlation among T3/TR,miR-21, and TIAM1 in vivo. A, theexpression of miR-21 from livertissues of SD rats underwent sham(n ¼ 4), thyroidetomies (Tx, n ¼ 4),and application of T3 (Tx þ T3,n ¼ 3) was detected using stem-loop qRT-PCR. B, the expressionof miR-21 from lung tissues of J7-TRa1 xenograft SCID mice witheu-, hypo-, and hyperthyroid(n¼ 3 in each group). C, lung tissuesections fromBwere carried out byH&E staining (I, IV, and VII:�200; II,V, and VIII: �400), andimmunohistochmistry wasdetected with TIAM1 (III, VI, and IX:�400). T, tumor. The black linesindicate that tumor region ofthe �400 magnification is from thecorresponding area of the�200. D,lung tissue section is fromcontrol SK-Hep1 xenografts(scrambled) and SK-Hep1miR-21 knockdown (anti-miR-21)xenografts. I and III displayed theH&E staining. II and IV displayedthe TIAM1 expression detected byimmunohistochemistry. Tumor isindicated by arrowheads. E, themetastatic index in lung tissue isshown. �, P < 0.05; ��, P < 0.01.

Huang et al.





TIAM1 suppression and upregulation of b-catenin, vimentin,andMMP2 (Supplementary Fig. S8).Moreover, the correlationsamong TRa1, miR-21, and TIAM1 expression patterns inpatients with hepatoma appear similar to those reported inhepatoma cells.Previous studies have reported that T3 stimulation affects

the expression of specific miRNAs in mouse hepatocyte cells.T3 upregulates Gpd2, andMup1mRNAmay be associated withdecreased miR-206 expression in the presence of T3 (38).However, the molecular mechanism underlying T3-mediatedregulation of miRNAs remains unclear at present. To ourknowledge, this is the first study to clearly show that themiR-21 promoter region containing native TRE bound tothyroid hormone receptor and RXR is directly upregulated byT3 in hepatoma cells. Both T3/thyroid hormone receptorsignaling and miR-21 stimulation promoted hepatoma cellmigration. Moreover, T3 and miR-21 stimulation was associ-ated with suppression of TIAM1 protein expression. Ourexperiments further confirmed that TIAM1 is a direct targetgene of miR-21. Notably, suppression of TIAM1 by T3 wasrescued upon knockdown of endogenousmiR-21. Suppressionof cell migration with anti-miR-21 was partially restored afteradministration of T3. Conversely, T3-enhanced cell migrationwas inhibited upon endogenous miR-21 knockdown. Overall,our analytic data support the promotion of hepatoma cellmigration and invasion through a T3/TR-miR-21-TIAM1 path-way (Supplementary Fig. S8). In addition, the tumor suppressorgene, MSH2 (39), another miR-21 target, was downregulatedthrough T3/thyroid hormone receptor signaling in hepatomacells. The associations among T3/thyroid hormone receptor,miR-21, and MSH2 further confirm the pathway of T3/TR-miR-21-target in hepatoma cells. On the basis of the reportedfunction of MSH2 as a tumor suppressor, our data supporta role of T3/thyroid hormone receptor as an oncogene.Suppression of TIAM1 expression by miR-21 or knockdown

of endogenous TIAM1 with RNAi led to the promotion ofmigration and invasion of Hep3B cells. Similar results wereobserved with colon carcinoma cells overexpressing miR-21,whereby enhanced cell migration and invasion were inhibiteduponTIAM1 overexpression (23). In addition, TIAM1 is a directtarget ofmiR-21 in LIM 1863 colon carcinoma cells (23). TIAM1inhibits the migratory and invasive properties of epithelial andmelanoma cells via different mechanisms. In epithelial cells,TIAM1 promotes E-cadherin–mediated cell–cell adhesion andshifts the balance between invasion-promoting MMP2 andMMP9 and invasion-inhibiting tissue inhibitors of metallopro-teinases (TIMP-1 and TIMP-2) to suppress cell migration andinvasion ability (40). In melanoma cells, TIAM1-mediatedinhibition ofmigration and invasion is dependent on increasedRac activity (41). In the current study, we propose that knock-down of TIAM1 promotes migratory and invasive abilities inhepatoma cells through activation of the b-catenin pathwayand increase in vimentin and MMP2 levels.Similarly, the estrogen receptor a (ERa) binds the miR-221-

222 transcription start site recruiting co-repressors to suppresstheir transcriptional activity (42). Interestingly, Zhao andcolleagues (43) reported that ERa is negatively regulated bymiR-221 andmiR-222 in breast cancer. These 2 studies support

the existence of a negative regulatory loop involving miR-221-222 and ERa (42). However, the issue of whether such aregulatory loop can be identified between miRNA and thyroidhormone receptor is yet to be established. In fact, degradationof thyroid hormone receptor induced via T3 binding has beenreported previously (44). In view of the finding that miR-27ainduces a decrease in TRb1 protein without affecting themRNA level (45), the possibility of amiR-21–triggered decreasein thyroid hormone receptor is speculated.

Wnt/b-catenin signaling is commonly activated in hepato-ma (46). Notably, hepatitis B virus X protein is reported toactivate b-catenin signaling (47). Furthermore, hepatitis Cvirus-induced miR-155 expression promotes hepatocyte pro-liferation and tumorigenesis via activation of Wnt signaling(48). Consistently, Lan and colleagues (49) observed a positivecorrelation between activation of the b-catenin pathway and insitu expression of miR-21 in colon carcinoma cells. In thecurrent study, we have shown that b-catenin signaling isactivated by miR-21, which also reported to be overexpressedin hepatoma (50) and upregulated by T3/thyroid hormonereceptor signaling.

In conclusion, our findings collectively illustrate thatmiR-21is directly upregulated by T3 through thyroid hormone recep-tor binding to native TRE of the miR-21 promoter region inhepatoma cells. T3 enhances migration and invasion by target-ing miR-21 to downregulate TIAM1 in hepatoma cells (Sup-plementary Fig. S8).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y.H. Huang, Y.H. Lin, H.C. Chi, C.J. Wang, K.H. LinDevelopment of methodology: Y.H. Lin, C.Y. Chen, C.J. Wang, K.H. LinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):Y.H. Huang, C.H. Liao, Y.H. Tseng, C.Y. Tsai, Y.T. Hung,C.J. Wang, C.D. Lin, K.H. LinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.H. Huang, C.H. Liao, Y.H. Tseng, C.Y. Tsai, S.Y. Lin,Y.T. Hung, C.J. Wang, K.H. LinWriting, review, and/or revision of the manuscript: Y.H. Huang, C.J. Wang,C.D. Lin, K.H. LinAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.J. Liao, S.M. Wu, C.J. Wang, K.H. LinStudy supervision: Y.H. Huang, C.J. Wang, K.H. Lin

AcknowledgmentsThe authors thank TLCN for providing the hepatoma tissue samples and

related clinical data (all are anonymous) for our research work. This networkcurrently includes 5majormedical centers (National TaiwanUniversity Hospital,Chang-Gung Memorial Hospital—Linko, Veteran General Hospital—-Taichung,Chang-Gung Memorial Hospital—Kaohsiung, and Veteran General Hospital—Kaohsiung). The authors also thank Hua-Chien Chen for the miRNAs initialscreening.

Grant SupportThis work was supported by grants from Chang-Gung University (Taoyuan,

Taiwan; grant no. CMRPD 34013, NMRP 140511) and from the National ScienceCouncil of the Republic of China (NSC 94-2320-B-182-052, NSC 98-2312-B-182A-001-MY3). TLCN is supported by grants fromNational ScienceCouncil since 2005till now (NSC 100-2325-B-182-006) and National Health Research Institutes,Taiwan.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 9, 2012; revised January 4, 2013; accepted February 4, 2013;published OnlineFirst February 26, 2013.






References1. Barlow C, Meister B, Lardelli M, Lendahl U, Vennstrom B. Thyroid

abnormalities and hepatocellular carcinoma in mice transgenic for v-erbA. EMBO J 1994;13:4241–50.

2. Gonzalez-Sancho JM, Garcia V, Bonilla F, Munoz A. Thyroid hormonereceptors/THRgenes in human cancer. Cancer Lett 2003;192:121–32.

3. Zamore PD, Haley B. Ribo-gnome: the big world of small RNAs.Science 2005;309:1519–24.

4. Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S,et al. HumanmicroRNAgenes are frequently locatedat fragile sites andgenomic regions involved in cancers. Proc Natl Acad Sci U S A2004;101:2999–3004.

5. Gregory RI, Shiekhattar R. MicroRNA biogenesis and cancer. CancerRes 2005;65:3509–12.

6. Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD, et al.MicroRNA profiling reveals distinct signatures in B cell chroniclymphocytic leukemias. Proc Natl Acad Sci U S A 2004;101:11755–60.

7. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, et al. AmicroRNA expression signature of human solid tumors defines cancergene targets. Proc Natl Acad Sci U S A 2006;103:2257–61.

8. Liao CH, Yeh SC, Huang YH, Chen RN, Tsai MM, Chen WJ, et al.Positive regulation of spondin 2 by thyroid hormone is associatedwith cell migration and invasion. Endocr Relat Cancer 2010;17:99–111.

9. Wu SM, Huang YH, Lu YH, Chien LF, Yeh CT, Tsai MM, et al. Thyroidhormone receptor-mediated regulation of the methionine adenosyl-transferase 1 gene is associatedwith cell invasion in cell lines. Cell MolLife Sci 2010;67:1831–43.

10. Chen RN, Huang YH, Lin YC, Yeh CT, Liang Y, Chen SL, et al. Thyroidhormone promotes cell invasion through activation of furin expressionin human hepatoma cell lines. Endocrinology 2008;149:3817–31.

11. Xu C, Liu S, Fu H, Li S, Tie Y, Zhu J, et al. MicroRNA-193b regulatesproliferation, migration and invasion in human hepatocellular carcino-ma cells. Eur J Cancer 2010;46:2828–36.

12. Huang YH, Lin KH, Chen HC, Chang ML, Hsu CW, Lai MW, et al.Identification of postoperative prognostic microRNA predictors inhepatocellular carcinoma. PLoS ONE 2012;7:e37188.

13. Chen CJ, Tsai NM, Liu YC, Ho LI, Hsieh HF, Yen CY, et al. Telomeraseactivity in human hepatocellular carcinoma: parallel correlation withhuman telomerase reverse transcriptase (hTERT) mRNA isoformexpression but not with cell cycle modulators or c-Myc expression.Eur J Surg Oncol 2002;28:225–34.

14. Huang YH, Liao CH, Chen RN, Liao CJ, Lin KH. Human testicularorphan receptor 4 enhances thyroid hormone receptor signaling. JCellPhysiol 2010;222:347–56.

15. Samuels HH, Stanley F, Casanova J. Depletion of L-3,5,30-triiodothy-ronine and L-thyroxine in euthyroid calf serum for use in cell culturestudies of the action of thyroid hormone. Endocrinology 1979;105:80–5.

16. Tai PJ, Huang YH, Shih CH, Chen RN, Chen CD, ChenWJ, et al. Directregulation of androgen receptor-associated protein 70 by thyroidhormone and its receptors. Endocrinology 2007;148:3485–95.

17. Huang YH, Lin KH, Liao CH, Lai MW, Tseng YH, Yeh CT. Furinoverexpression suppresses tumor growth and predicts a better post-operative disease-free survival in hepatocellular carcinoma. PLoSONE 2012;7:e40738.

18. Chen CY, Chi LM, Chi HC, Tsai MM, Tsai CY, Tseng YH, et al. Stableisotope labeling with amino acids in cell culture (SILAC)-based quan-titative proteomics study of a thyroid hormone-regulated secretome inhuman hepatoma cells. Mol Cell Proteomics 2012;11:M111 011270.

19. Chi HC, Chen SL, Liao CJ, Liao CH, Tsai MM, Lin YH, et al. Thyroidhormone receptors promote metastasis of human hepatoma cells viaregulation of TRAIL. Cell Death Differ 2012;19:1802–14.

20. Medina PP, Nolde M, Slack FJ. OncomiR addiction in an in vivomodelof microRNA-21-induced pre-B-cell lymphoma. Nature 2010;467:86–90.

21. Chen RN, Huang YH, Yeh CT, Liao CH, Lin KH. Thyroid hormonereceptors suppress pituitary tumor transforming gene 1 activity inhepatoma. Cancer Res 2008;68:1697–706.

22. LiaoCH,YehCT,HuangYH,WuSM,ChiHC, TsaiMM, et al. Dickkopf 4positively regulated by the thyroid hormone receptor suppresses cellinvasion in human hepatoma cells. Hepatology 2012;55:910–20.

23. Cottonham CL, Kaneko S, Xu L. miR-21 and miR-31 converge onTIAM1 to regulate migration and invasion of colon carcinoma cells. JBiol Chem 2010;285:35293–302.

24. Gao W, Xu J, Liu L, Shen H, Zeng H, Shu Y. A systematic-analysis ofpredictedmiR-21 targets identifiesa signature for lung cancer. BiomedPharmacother 2012;66:21–8.

25. Kim YJ, Hwang SJ, Bae YC, Jung JS. MiR-21 regulates adipogenicdifferentiation through the modulation of TGF-beta signaling in mes-enchymal stem cells derived from human adipose tissue. Stem Cells2009;27:3093–102.

26. YangX,Wang J,GuoSL, FanKJ, Li J,WangYL, et al.miR-21 promoteskeratinocyte migration and re-epithelialization during wound healing.Int J Biol Sci 2011;7:685–90.

27. Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY. MicroRNA-21 targetstumor suppressor genes in invasion andmetastasis. Cell Res 2008;18:350–9.

28. Zhu Q, Wang Z, Hu Y, Li J, Li X, Zhou L, et al. miR-21 promotesmigration and invasion by the miR-21-PDCD4-AP-1 feedbackloop in human hepatocellular carcinoma. Oncol Rep 2012;27:1660–8.

29. AdamsHC III, ChenR, Liu Z,Whitehead IP.Regulation of breast cancercell motility by T-cell lymphoma invasion and metastasis-inducingprotein. Breast Cancer Res 2010;12:R69.

30. Connolly EC, Van Doorslaer K, Rogler LE, Rogler CE. Overexpressionof miR-21 promotes an in vitro metastatic phenotype by targeting thetumor suppressor RHOB. Mol Cancer Res 2010;8:691–700.

31. Birchmeier C, Birchmeier W, Brand-Saberi B. Epithelial-mesenchymaltransitions in cancer progression. Acta Anat 1996;156:217–26.

32. Hu L, Lau SH, Tzang CH,Wen JM,WangW, Xie D, et al. Association ofvimentin overexpression and hepatocellular carcinoma metastasis.Oncogene 2004;23:298–302.

33. Lai TY, Su CC, Kuo WW, Yeh YL, Kuo WH, Tsai FJ, et al. beta-cateninplays a key role in metastasis of human hepatocellular carcinoma.Oncol Rep 2011;26:415–22.

34. Chen PS, Su JL, Cha ST, Tarn WY, Wang MY, Hsu HC, et al. miR-107promotes tumor progression by targeting the let-7 microRNA in miceand humans. J Clin Invest 2011;121:3442–55.

35. Vaksman O, Stavnes HT, Kaern J, Trope CG, Davidson B, Reich R.miRNA profiling along tumour progression in ovarian carcinoma. J CellMol Med 2011;15:1593–602.

36. Szczyrba J, Loprich E, Wach S, Jung V, Unteregger G, Barth S, et al.The microRNA profile of prostate carcinoma obtained by deepsequencing. Mol Cancer Res 2010;8:529–38.

37. Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T,et al. Comprehensive analysis of microRNA expression patterns inhepatocellular carcinoma and non-tumorous tissues. Oncogene2006;25:2537–45.

38. Dong H, Paquette M, Williams A, Zoeller RT, WadeM, Yauk C. Thyroidhormone may regulate mRNA abundance in liver by acting on micro-RNAs. PLoS ONE 2010;5:e12136.

39. Zink D, Mayr C, Janz C, Wiesmuller L. Association of p53 and MSH2with recombinative repair complexes during S phase. Oncogene2002;21:4788–800.

40. Macharashvili N, Kourbatova E, Butsashvili M, Tsertsvadze T, McNuttLA, LeonardMK. Etiology of neonatal blood stream infections in Tbilisi,Republic of Georgia. Int J Infect Dis 2009;13:499–505.

41. Uhlenbrock K, Eberth A, HerbrandU, Daryab N, Stege P, Meier F, et al.The RacGEF TIAM1 inhibits migration and invasion of metastaticmelanoma via a novel adhesive mechanism. J Cell Sci 2004;117:4863–71.

42. Di Leva G, Gasparini P, Piovan C, Ngankeu A, Garofalo M, TaccioliC, et al. MicroRNA cluster 221-222 and estrogen receptor alphainteractions in breast cancer. J Natl Cancer Inst 2010;102:706–21.

43. Zhao JJ, Lin J, Yang H, Kong W, He L, Ma X, et al. MicroRNA-221/222negatively regulates estrogen receptor alpha and is associated

Huang et al.





with tamoxifen resistance in breast cancer. J Biol Chem 2008;283:31079–86.

44. Chen SL, Chang YJ, Wu YH, Lin KH. Mitogen-activated proteinkinases potentiate thyroid hormone receptor transcriptionalactivity by stabilizing its protein. Endocrinology 2003;144:1407–19.

45. Nishi H, Ono K, Horie T, Nagao K, Kinoshita M, Kuwabara Y, et al.MicroRNA-27a regulates beta cardiac myosin heavy chain geneexpression by targeting thyroid hormone receptor beta1 in neonatalrat ventricular myocytes. Mol Cell Biol 2011;31:744–55.

46. Lachenmayer A, Alsinet C, Savic R, Cabellos L, Toffanin S, Hoshida Y,et al. Wnt-pathway activation in two molecular classes of hepatocel-lular carcinoma and experimental modulation by sorafenib. Clin Can-cer Res 2012;18:4997–5007.

47. Srisuttee R, Koh SS, Kim SJ, Malilas W, Boonying W, Cho IR, et al.Hepatitis B virus X (HBX) protein upregulates beta-catenin in a humanhepatic cell line by sequestering SIRT1 deacetylase. Oncol Rep2012;28:276–82.

48. Zhang Y, Wei W, Cheng N, Wang K, Li B, Jiang X, et al. Hepatitis Cvirus-induced up-regulation of microRNA-155 promotes hepatocarci-nogenesis by activating Wnt signaling. Hepatology 2012;56:1631–40.

49. Lan F, Yue X, Han L, Shi Z, Yang Y, Pu P, et al. Genome-wideidentification of TCF7L2/TCF4 target miRNAs reveals a role for miR-21 in Wnt-driven epithelial cancer. Int J Oncol 2012;40:519–26.

50. Ladeiro Y, Couchy G, Balabaud C, Bioulac-Sage P, Pelletier L,Rebouissou S, et al. MicroRNA profiling in hepatocellular tumors isassociated with clinical features and oncogene/tumor suppressorgene mutations. Hepatology 2008;47:1955–63.






2013;73:2505-2517. Published OnlineFirst February 26, 2013.Cancer Res Ya-Hui Huang, Yang-Hsiang Lin, Hsiang-Cheng Chi, et al. Invasion of HepatomaThyroid Hormone Regulation of miR-21 Enhances Migration and

Updated version

10.1158/0008-5472.CAN-12-2218doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2013/02/26/0008-5472.CAN-12-2218.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/73/8/2505.full#ref-list-1

This article cites 50 articles, 14 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/73/8/2505.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/73/8/2505To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-12-2218

http://cancerres.aacrjournals.org/content/suppl/2013/02/26/0008-5472.CAN-12-2218.DC1

http://cancerres.aacrjournals.org/content/73/8/2505.full#ref-list-1

http://cancerres.aacrjournals.org/content/73/8/2505.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/73/8/2505


thyroid hormone regulation of mir-21 enhances migration ... · molecular and cellular pathobiology...

Documents