Functional Role of a Novel Long Noncoding RNA in ... · Esophageal carcinoma is the sixth leading cause of tumor-related mortality worldwide (1). There are two main esophageal carcinoma

Biology of Human Tumors

Functional Role of a Novel Long Noncoding RNATTN-AS1 in Esophageal Squamous Cell CarcinomaProgression and MetastasisChenyu Lin1,2, Shengnan Zhang1,2, Ying Wang1,2, Yuanshu Wang1,2,Edouard Nice3, Changying Guo1,2, Erhao Zhang1,2, Liting Yu1,2, Mengwei Li1,2,Chen Liu1,2, Lirong Hu1,2, Jingchao Hao1,2,4,Weiyan Qi1,2, and Hanmei Xu1,2

Abstract

Purpose: Emerging studies demonstrate that long noncodingRNAs (lncRNA) participate in the regulation of various cancers. Inthe current study, a novel lncRNA-TTN-AS1 has been identifiedand explored in esophageal squamous cell carcinoma (ESCC).

Experimental Design: To discover a new regulatory circuitry inwhich RNAs crosstalk with each other, the transcriptome oflncRNA-miRNA-mRNA from ESCC and adjacent nonmalignantspecimens were analyzed using multiple microarrays and diversebioinformatics platforms. The functional role and mechanism ofa novel lncRNA-TTN-AS1 were further investigated by gain-of-function and loss-of-function assays in vivo and in vitro. An ESCCbiomarker panel, consisting of lncRNA-TTN-AS1, miR-133b, andFSCN1, was validated by qRT-PCR and in situ hybridization usingsamples from 148 patients.

Results: lncRNA-TTN-AS1 as an oncogene is highly expressedin ESCC tissues and cell lines, and promotes ESCC cell pro-

liferation and metastasis. Mechanistically, lncRNA-TTN-AS1promotes expression of transcription factor Snail1 by compet-itively binding miR-133b, resulting in the epithelial–mesenchy-mal transition (EMT) cascade. Moreover, lncRNA-TTN-AS1 alsoinduces FSCN1 expression by sponging miR-133b and upregu-lation of mRNA-stabilizing protein HuR, which further pro-motes ESCC invasion cascades. We also discovered and vali-dated a clinically applicable ESCC biomarker panel, consistingof lncRNA-TTN-AS1, miR-133b, and FSCN1, that is significantlyassociated with overall survival and provides additional prog-nostic evidence for ESCC patients.

Conclusions: As a novel regulator, lncRNA-TTN-AS1 plays animportant role in ESCC cell proliferation and metastasis. ThelncRNA-TTN-AS1/miR133b/FSCN1 regulatory axis provides bonafide targets for anti-ESCC therapies. Clin Cancer Res; 24(2); 486–98.�2017 AACR.

IntroductionEsophageal carcinoma is the sixth leading cause of tumor-

related mortality worldwide (1). There are two main esophagealcarcinoma types: esophageal squamous cell carcinoma (ESCC)and adenocarcinoma (EAC). ESCC is the predominant subtype ofesophageal carcinoma in Asia. Although multimodal therapieshave improved treatment and prognosis of esophageal carcino-ma, the overall 5-year survival rate is still poor (2). The pooroutcomes of ESCC are associated with diagnosis at advanced

stages and the propensity for metastasis (3). Therefore, diagnosisof ESCC in the early stages is crucial.

With advances in high throughput analysis, increasing tumor-related noncoding RNAs have been identified (4). Particularly,accumulating esophageal carcinoma-related long noncodingRNAs (lncRNAs) have been verified to exert diverse functionsthrough various biological processes. For example, HNF1A-AS1induces H19 expression and modulates chromatin and nucleo-some assembly, resulting in gene imprinting (5). HOX transcriptantisense RNA (HOTAIR) suppressesWIF-1 expression by induc-ing histone H3K27 methylation in the promoter region (6).Numerous esophageal carcinoma-related lncRNAs have beenfound, but the precise molecular mechanisms of most lncRNAsin ESCC are still not fully understood.

In this study, we identified a novel lncRNA-TTN-AS1(ENST00000589434) and demonstrated that high levels oflncRNA-TTN-AS1 were correlated with poor ESCC prognosis,tumor growth, and invasion cascades. Furthermechanistic studiesrevealed that lncRNA-TTN-AS1 upregulates Snail1 and actin-binding protein fascin homolog 1 (FSCN1) by competitive reg-ulationofmiR-133b, resulting inESCCcellmetastasis. In addition,lncRNA-TTN-AS1 also facilitates and combines directly withthe HuR to stabilize FSCN1 mRNA. Taken together, the studyunveils a novel biomarker panel, consisting of lncRNA-TTN-AS1/miR133b/FSCN1, which plays a pivotal role in ESCC progres-sion and metastasis.

1Department of Engineering Research Center of Peptide Drug Discovery andDevelopment, Nanjing, P.R. China. 2Department of State Key Laboratory ofNatural Medicines, China Pharmaceutical University, Nanjing, P.R. China.3Department of Biochemistry and Molecular Biology, Monash University, Clay-ton, Victoria, Australia. 4School of Pharmacy and The Yunnan Provincial KeyLaboratory of Natural Drug and Pharmacology, Kunming Medical University,Kunming, Yunnan, P.R. China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

C. Lin and S. Zhang share co-first authorship of this article.

Correspondence Author: Hanmei Xu, China Pharmaceutical University, Nanjing210009, China. Phone: 8613-9139-25346; Fax: 8625-8327-1007; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-17-1851

�2017 American Association for Cancer Research.

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Materials and MethodsPatients and specimens

Two independent cohorts comprising 148 ESCC patients wereenrolled for this study. In cohort 1, ESCC and adjacent nontumorspecimens were gathered from 58 patients who were diagnosedwith ESCC between December 2014 and November 2015 atNanjing General Hospital of Nanjing Military Command(Jiangsu, China). The study was approved by Nanjing GeneralHospital of Nanjing Military Command Review Board, and writ-ten informed consent was obtained from all participants. Incohort 2, paraffin-embedded tissue samples were collected fromarchival material stored in the Biobank Center at the NationalEngineering Center for Biochip at Shanghai (Shanghai OutdoBiotech Co., Ltd.). Specimens from ESCC and adjacent nonma-lignant tissues were collected from 90 ESCC patients who under-went surgical resection between 2006 and 2008, and were fol-lowed up for 7.8 years. The clinical characteristics of all patientsare listed in Supplementary Tables S4 and S7. All patients werediagnosed according to the guidelines of the American JointCommission on Cancer and the guidelines of the InternationalUnion Against Cancer (IUAC). The study was conducted inaccordance with Ethics Committee of China PharmaceuticalUniversity.

lncRNAþmRNA microarraySeven paired samples of ESCC versus adjacent noncancer-

ous tissues were selected for microarrays analysis (OutdoBiotech Co., Ltd.; Supporting Information Table S1). Briefly,fluorescent (Cy5 and Cy3-dCTP) labeled cDNA was synthe-sized and hybridized to the 4 � 180 K Agilent humanlncRNAþmRNA Array v4.0 (Agilent). After hybridization andwashing, the slides were scanned using an Agilent G2565CAMicroarray Scanner. Quantile normalization and differentialanalysis were performed with the Agilent GeneSpring softwarev.11.5 (Agilent Technologies Inc.). To select the differentiallyexpressed genes, we used threshold values of �2 and ��2fold change and a Benjamini–Hochberg corrected P value of0.05. The data were log2 transformed and median centered bygenes using the Adjust Data function of CLUSTER 3.0 software(University of Tokyo, Human Genome Center, Tokyo, Japan)and then further analyzed with hierarchical clustering withaverage linkage. Finally, we performed tree visualization byusing Java TreeView (Stanford University School of Medicine,Stanford, CA).

miRNA microarraymiRNA microarray analysis was undertaken using the above

seven paired samples of ESCC versus adjacent noncanceroustissues (Outdo Biotech Co., Ltd.). Briefly, miRNAs were extractedand purified from total RNAusing amirVanamiRNA Isolation Kit(Ambion), then a poly A tail was added in the 30 end of miRNAusing poly A polymerase and labeled with biotin labeling 3DNAdendrimer (FlashTag Biotin RNA Labeling). Biotin-labeledmiRNA hybridized to the Affymetrix GeneChip miRNA Array4.0. Array (Affymetrix) was scanned with a GeneChip Scanner3000, and the images processed using the AGCC software (Affy-metrix GeneChip Command Console Software). Signals werenormalized by the median center tool for genes in the Cluster3.0 software and analyzed by significance analysis of microarrays(SAM), with the FDR threshold set at 0 and fold change set (foldchange �2; P � 0.05 or change � �2; P � 0.05).

Cell culture and reagentsESCC cell lines Eca-109, KYSE 30, KYSE 150, KYSE180,

KYSE410, KYSE450, KYSE510, TE-10, and TE-13 and normalesophageal epithelial cell lines HECC kindly supplied by Profes-sor Yifeng Zhou, Suzhou University in 2015. The cell lines havebeen tested and authenticated by the company. All cells weretested for mycoplasma every 3 months and were negative. Anti-bodies against cleaved-caspase-3, cleaved-caspase-9, HuR, b-cate-nin, Lamin B1, and Ki-67 were purchased from Cell SignalingTechnology. Antibodies against pro-caspase-3, pro-caspase 9,b-actin, Bcl-2, Bax, E-cadherin, ZO-1, N-cadherin, Vimentin,Snail1, Argonaute2 (Ago2), and FSCN1 were purchased fromAbcam. Flag antibody was purchased from Sigma-Aldrich. Cellviability assay was performed using CCK8 (Dojindo) kits. Pro-pidium Iodide and Annexin V were purchased from Biolegend.

Cell viability, colony formation, and in vivo xenograft assaysThe cell viability, colony formation, and in vivo tumor growth

assayswere performed as described previously (7), and as detailedin the Supplementary Materials and Methods section.

Quantitative real-time PCR, Western blot analysis, and flowcytometry

The procedures for performing qRT-PCR and Western blotanalysis have been described previously (7). Flow cytometry wasconducted according to the manufacturer's standard protocol.

AnimalsFemale Balb/c nudemice (aged, 4–5weeks; Cavens Lab Animal

Co.) were cared for according to Provisions and General Recom-mendation of Chinese Experimental Animals AdministrationLegislation. The procedure of all animal experiments compliedwith Institutional Animal Care and Use Committee (IACUC)regulations. All animal experiments were approved by the EthicsCommittee of China Pharmaceutical University Permit Number:SYXK2012-0035.

Transwellmigration/invasion andwound healing assays and invivo imaging assays

The migration/invasion assays, wound healing assays and invivo tumor growth and imaging assaywere performed as describedpreviously (7), and as detailed in the Supplementary Materialsand Methods section.

Translational Relevance

Long noncoding RNAs (lncRNA) play pivotal roles inesophageal squamous cell carcinoma (ESCC) proliferation,metastasis, diagnosis, and prognosis. We identified a novelESCC-related lncRNA-TTN-AS1 as a vital regulator of ESCCprogression. lncRNA-TTN-AS1 promoted snail1 and FSCN1expression by competitively binding miR-133b and inter-acting with mRNA to stabilize protein HuR, resulting inactivation of a metastasis cascade. The biomarker panel oflncRNA-TTN-AS1-miR-133b-FSCN1 correlateswith overall sur-vival and provides accurate prognostic evidence.

lncRNA-TTN-AS1 Facilitates ESCC Progression

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Dual luciferase reporter assaysLuciferase activities were performed using the Dual Luciferase

Assay Kit (Promega) according to themanufacturer's instructions.

RNA pull-down and RNA immunoprecipitation assaysRNA pull down and RNA immunoprecipitation (RIP) were

performed as described previously (7, 8). For RNA pull downassay, RNAs were biotin-labeled and in vitro transcribed withBiotin RNA Labeling Mix (Roche) and T7/SP6 RNA polymerase(Roche). Cell lysates were mixed and incubated with biotinylatedRNAs. Streptavidin agarose beads were added to each bindingreaction, followed by 1-hour incubation at room temperature.Beads were washed and boiled in SDS buffer, and the retrievedproteins detected by Western blot analysis.

The Magna RIP Kit (Millipore) was used in RIP experimentsaccording to the manufacturer's instructions. The coprecipitatedRNAs were detected by qRT-PCR.

RNA-LNA in situ hybridizationIn situ hybridization (ISH) of lncRNA/mRNA and miRNA with

ESCC tissue microarrays (TMA) were performed by ShanghaiOutdo Biotech Co., Ltd. (catalog no. HEso-Squ180Sur-04). Forthe TMAs, there were 90 ESCC patient samples complete withsurvival times and related clinicopathologic characteristics. Thetissue array was stained with hematoxylin and eosin (H&E) toverify the presence of tumor cells. Detailed descriptions can befound in the Supplementary Materials and Methods section.

Statistical analysisStatistical analyses were performed using SPSS statistics 22.0.

The paired t test was performed to detect the differential expres-sion of lncRNA-TTNF-AS1 in ESCC cancer tissues compared withadjacent normal tissues. The relationship between lncRNA-TTN-AS1 and clinicopathologic characteristics was evaluated using c2

test. Survival curves were calculated using Kaplan–Meier and log-rank tests. The effects of variables on survival were analyzed byunivariate and multivariate Cox proportional hazards modeling.For two group comparison, multiple group comparison andcorrelation analyses were calculated with a paired two-tailedStudent t test, two-way ANOVA test, linear regression test, andPearson test using GraphPad Prism 5 software (Graph Pad soft-ware Inc.), respectively. P values less than 0.05 were consideredstatistically different (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

ResultsUpregulation of lncRNA-TTN-AS1 in ESCC tissues and cell lines

To understand the regulatory circuitries by which RNAs cancrosstalk with each other, the expression profiles of lncRNA,mRNA, and miRNA in ESCC tissues and adjacent normal tissuesfrom seven ESCC patients (Supplementary Table S1) weredetected by multiple microarrays. Comparison of differentlyexpressed miRNAs was calculated by single channel chip andnormalized by Lowess (Fig. 1A). lncRNA and mRNA expressionprofiles were detected by dual channel chip (Fig. 1B and C). Allhierarchical clustering results showed systematic variation intranscript levels between ESCC tissues and adjacent normaltissues. The microarray data have been deposited in NCBI GeneExpression Omnibus and are accessible through GEO SeriesAccession Number GSE97051 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE97051). To validate our microarray

data, 14 transcripts with significantly different expression wereexamined by qRT-PCR (Supplementary Fig. S1). The resultsshowed that the levels of 11 transcripts were consistent with themicroarray data (Supplementary Fig. S4A). Furthermore, poten-tial lncRNAs were screened using coexpression network (Fig. 1D)and diverse bioinformatics tools (Fig. 1E). Finally, the number ofmiRNA–lncRNA pairs with significant correlation was narrowedto three (Supplementary Table S3). Microarray data showedENST00000589434 was notably downregulated in ESCC tissuesand the correlation score between ENST00000589434 and hsa-miR-133b was the highest. Therefore, we focused on the functionand mechanism of lncRNA-TTN-AS1 (ENST00000589434) inESCC. Notably, lncRNA-TTN-AS1 is derived from the oppositestrand of Titin (TTN) gene, which encodes a large abundantprotein of striated muscle, meanwhile, miR-133b is derived fromlinc-MD1, which modulates early phases of muscle differentia-tion. Hence, we also detected the expression of TTN and linc-MD1in ESCC specimens, as shown in Supplementary Fig. S4B andS4C,TTN and linc-MD1 levels betweenESCCandadjacent normaltissues were significantly different.

The potential coding capability of lncRNA-TTN-AS1 was alsoevaluated as follows: although two short reading frames (ORF3and ORF5) with more than 200 nt were predicted using ORFFinder from the National Center for Biotechnology Information(Supplementary Fig. S2A), neither of their AUGs showed theKozak consensus, nor were homologous protein sequences foundusing a BLAST search; PhyloCSF value of all the exons of lncRNA-TTN-AS1 were less than zero and their sequences were lessconserved, which further suggested that it was unlikely to encodeany protein (Supplementary Fig. S2B); the online bioinformaticsanalysis (coding potential calculator) also confirmed lncRNA-TTN-AS1 has no coding capability (coding potential score:�1.11259; http://cpc.cbi.pku.edu.cn/programs/run_cpc.jsp) inagreement with our finding that lncRNA-TTN-AS1 has no codingcapability (Supplementary Fig. S2C).

lncRNA-TTN-AS1 regulates miR-133b as a ceRNAIt is well known that noncoding RNAs as competing endoge-

nous RNA (ceRNA) bind tomiRNAs and protect their target RNAsfrom repression or degradation. The downregulation ofmiR-133bthat was found in human ESCC (9) prompted us to see whetherlncRNA-TTN-AS1 was negatively correlated with miR-133b inESCC tissues. As expected, lncRNA-TTN-AS1 was robustly upre-gulated in ESCC tissues in cohort 1 (P ¼ 0.000; SupplementaryFig. S3A.). Conversely, miR-133b expression was significantlydownregulated in ESCC tissues (P ¼ 0.000; Supplementary Fig.S3B).Moreover, ISH studies also confirmed that lncRNA-TTN-AS1significantly increased in ESCC (Fig. 6A). In addition, comparedto normal esophageal epithelial cells (HEEC) cells, lncRNA-TTN-AS1 expression was notably higher in ESCC cell lines, whereasmiR-133b level was lower (Supplementary Fig. S3C and S3D).Consistently, a strong negative correlation was found betweenlncRNA-TTN-AS1 andmiR-133b in ESCC tissues (r¼�0.8704, P <0.001; Supplementary Fig. S3E) and cell lines (r ¼ �0.8500, P <0.001; Supplementary Fig. S3F). Although the qRT-PCR resultswere not fully consistent with the microarray data, lncRNA-TTN-AS1 has been validated as a potential oncogene.

Next we performed luciferase reporter assays and RNA pull-down assays to test the direct binding between lncRNA-TTN-AS1and miR-133b. The luciferase intensity was decreased by cotrans-fected miR-133bmimics and lncRNA-TTN-AS1-WT but not in the

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Figure 1.

Screening of ESCC-related lncRNAs using multiple microarrays and bioinformatics. Hierarchical clustering analysis of significantly expressed miRNAs (A), lncRNAs(B), and mRNAs (C) in ESCC tissues and adjacent normal tissues (>2-fold; P < 0.05). (D) Coexpression network of miRNAs, lncRNAs, and mRNAs. Colorednodes, miRNAs; black nodes, lncRNAs; gray nodes, mRNAs; colored edges indicate miRNA–target interactions. mRNAs' names not shown. E,Diagrams of significantcorrelation of lncRNA–miRNA pairs predicted in silico.






mutant reporter vector lacking the putativemiR-133b binding site(Fig. 2A). Consistently, lncRNA-TTN-AS1 was pulled down bybiotin-labeled miR-133b, whereas miR-133b mutant could notpull down lncRNA-TTN-AS1. In a reciprocal manner, miR-133bwas also precipitated by wild-type lncRNA-TTN-AS1 but not thelncRNA-TTN-AS1 mutant (Fig. 2B). These data suggest thatlncRNA-TTN-AS1 is a bona fide miR-133b–targeting lncRNA.

Intriguingly, miR-133b was downregulated by lncRNA-TTN-AS1 overexpression, as well as upregulated by lncRNA-TTN-AS1knockdown (Supplementary Fig. S3I and S3J). However, nosignificant difference of lncRNA-TTN-AS1 expressionwas detectedafter ectopic expressionor deficiency ofmiR-133b (SupplementaryFig. S4D and S4E). Moreover, overexpression of lncRNA-TTN-AS1 significantly attenuated miR-133b level in contrast to themutant lacking the miR-133b targeting site (Fig. 2C). These dataconfirm that miR-133b binds lncRNA-TTN-AS1 without degrada-tion of lncRNA-TTN-AS1.

lncRNA-TTN-AS1 was mainly found in the cytoplasm of ESCCcell lines (Fig. 2D), which suggested that lncRNA-TTN-AS1 maybind to miR-133b through the Ago2-dependent RNAi pathway.As expected, RIP assay showed levels of lncRNA-TTN-AS1 andmiR-133b precipitated by anti-Ago2 antibody were markedlyincreased, with a 2- to 3-fold enrichment compared with IgG(Fig. 2E). Meanwhile, endogenous lncRNA-TTN-AS1 pull-downby Ago2 was specifically enriched upon overexpression of miR-133b (Fig. 2F). These data confirmed that lncRNA-TTN-AS1 wasbound to miR-133b in the cytoplasm in an Ago2-dependentmanner. To further confirm lncRNA-TTN-AS1 as a ceRNA, wecompared the abundance of lncRNA-TTN-AS1 andmiR-133b. Theexact copy numbers of lncRNA-TTN-AS1 (�1.45 copies per cell)was higher than that ofmiR-133b (�0.37 copies per cell) in TE-13cells (Supplementary Fig. S4F). Moreover, lncRNA-TTN-AS1 over-expression decreased the copy numbers ofmiR-133b (Supplemen-tary Fig. S4G). Taken together, these data indicate that lncRNA-TTN-AS1 physically interacts with miR-133b as a ceRNA.

The role of lncRNA-TTN-AS1 in ESCC cell proliferation, cellapoptosis, and cell-cycle progression

To dissect the effect of lncRNA-TTN-AS1 in ESCC progression,gain- and loss-of-function assays were performed using ESCC celllines. lncRNA-TTN-AS1 was inhibited in KYSE-410 cells and thenwe stably overexpressed miR-133b in lncRNA-TTN-AS1–overex-pressing clones (Supplementary Fig. S3G and S3I). Meanwhile,lncRNA-TTN-AS1 was stably silenced in TE-13 cells, followed byknockdown of miR-133b in lncRNA-TTN-AS1 deleted clones(Supplementary Fig. S3H and S3J).

To explore the influence of lncRNA-TTN-AS1 on ESCC prolif-eration, CCK-8 and colony formation assays were performed.Ectopic expression of lncRNA-TTN-AS1 induced cell proliferationand colony formation, whereas overexpression of miR-133babolished this increase (Fig. 3B; Supplementary Fig. S6A). In anin vivo assay, tumor growth in xenografts with lncRNA-TTN-AS1–overexpressing clones was increased compared with that of anegative control, whereas ectopic expression of miR-133b elimi-nated the lncRNA-TTN-AS1–induced tumor growth (Fig. 3D). Inaddition, overexpression of lncRNA-TTN-AS1 augmented theproportion of proliferating (Ki67þ) cancer cells (SupplementaryFig. S6C).

To further investigate the effect of lncRNA-TTN-AS1 andmiR133b on cell proliferation, apoptosis-related experimentswere performed. As shown in Fig. 3A, the percentage of Annexin

V-Light 650-positive cells decreased upon overexpression oflncRNA-TTN-AS1, whereas ectopic expression of miR-133b abro-gated the decrease. Consistently, overexpression of lncRNA-TTN-AS1 resulted in reduction of well-known apoptotic proteins,including cleaved caspase-3, cleaved caspase-9, and Bax andincrease of antiapoptosis protein Bcl-2 in ESCC cell lines thatwas overcome by ectopic expression of miR-133b (Fig. 3C).Furthermore, cell-cycle analysis confirmed ESCC cells with over-expressing lncRNA-TTN-AS1 had a significantly reduced G1 pop-ulation and amarkedly increased S-phase, and ectopic expressionofmiR-133b reversed the above phenomena (Supplementary Fig.S6B). Collectively, these results indicate that lncRNA-TTN-AS1induces cell proliferation by inactivation of apoptosis-relatedsignaling pathway and facilitates cell-cycle progression.

lncRNA-TTN-AS1 promotes Snail1 expressionBecause miR-133b targets Snail1 (10) and lncRNA-TTN-AS1

shares a miR-133b response element with Snail1, we reasonedthat lncRNA-TTN-AS1 could induce EMT-transcription factorSnail1 and promote invasion of ESCC cells. We found thatlncRNA-TTN-AS1 significantly increased Snail1 expression, whichcould be abrogated by ectopic expression of miR-133b (Supple-mentary Fig. S5A and S5B). Furthermore, dual luciferase reporterassays validated ectopic expression of lncRNA-TTN-AS1, but notthe mutant, increased the luciferase intensity, which could beabolished by overexpression of miR-133b (SupplementaryFig. S5D). In addition, lncRNA-TTN-AS1was positively correlatedwith Snail1 mRNA level (Supplementary Fig. S5E). These dataillustrate that lncRNA-TTN-AS1 modulates Snail1 by competi-tively binding miR-133b, which may further promote ESCCmetastasis.

lncRNA-TTN-AS1 induces ESCC cell metastasis in vitro andin vivo

To further examine the effect of lncRNA-TTN-AS1 on ESCC cellmetastasis and EMT cascades, cell migration and invasion andwound healing assays were conducted. Ectopic expression oflncRNA-TTN-AS1 promoted cell migration, cell invasion, andscratch closure rate, whereas overexpression of miR-133b attenu-ated lncRNA-TTN-AS1-induced cell metastasis (Fig. 4A; Supple-mentary Fig. S6D and S6E). Meanwhile, lncRNA-TTN-AS1 signif-icantly induced mesenchymal markers N-cadherin and Vimentinand decreased expression of epithelial markers E-cadherin andZO-1, which were rescued by ectopic expression of miR-133b(Fig. 4B and C). In addition, lncRNA-TTN-AS1 abolished therepression of Snail1 and EMT induced bymiR-133b (Supplemen-tary Fig. S7B and S7C). In summary, our data demonstrate thatlncRNA-TTN-AS1 plays a pivotal role in activating EMT by thecompetitive binding of miR-133b.

To ascertain the correlation between lncRNA-TTN-AS1 andEMT markers, we examined the levels of EMT markers in fourESCC cell lines with different expression of lncRNA-TTN-AS1.High levels of Snail1, N-cadherin, and Vimentin and low levels ofE-cadherin and ZO-1 were observed in lncRNA-TTN-AS1 highexpression cells (Supplementary Fig. S7D and S7E). Consistently,the lncRNA-TTN-AS1 transcript was negatively correlated withE-cadherin mRNA levels in ESCC specimens (SupplementaryFig. S5F).

To evaluate the effect of lncRNA-TTN-AS1 on tumor metastasisin vivo, we then intravenously injected the indicated ESCC cellsinto nude mice to establish a tumor metastasis model. In vivo

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Figure 2.

lncRNA-TTN-AS1 interacts with miR-133b in ESCC cell lines. A, Schematic map of constructs used in dual-luciferase reporter assays, as shown at left; luciferaseintensities in TE-13 cells cotransfected with miR-133b mimic or NC and luciferase reporters containing lncRNA-TTN-AS1 wild-type or mutant at right. B, RNApull-down assay using biotin-labeled miR-133b (left) or biotin-labeled lncRNA-TTN-AS1 (right) in TE-13 cells. C, Relative expression of miR-133b in TE-13 cellsupon overexpression of lncRNA-TTN-AS1 or mutant lacking the miR-133b binding site. D, Subcellular level of lncRNA-TTN-AS1 in ESCC cell lines. E, Relativeenrichment of lncRNA-TTN-AS1 and miR-133b in RIP using anti-Ago2 antibody in TE-13 cells. The fold enrichment of lncRNA-TTN-AS1 and miR-133bnormalized to nonspecific IgG as negative control. F, Ago2-associated lncRNA-TTN-AS1 in TE-13 cells with ectopic expression of miR-133b. lncRNA-TTN-AS1expression was normalized to b-actin level, andmiR-133b expression was normalized to U6 small RNA expression. Data were presented as mean � SD. �, P < 0.05;�� , P < 0.01; �� , P < 0.001, Student t test.






Figure 3.

lncRNA-TTN-AS1promotes cell proliferation and inhibits apoptosis signaling.A, The apoptotic cell percentagewas determined usingAnnexin V-Light 650 reagents inthe indicated ESCC cell lines. B, Cell viability was determined in indicated KYSE-410 cells (top) and TE-13 cells (bottom). C, Western blot analysisof cleaved caspase-3, pro-caspase, cleaved caspase-9, pro-caspase-9, Bcl-2, Bax, and b-actin control. D, The tumor growth of mice in each group followingsubcutaneous implantation with the indicated ESCC cell lines. Left, Representative images of tumors formed in nude mice; middle, tumor volume; right, tumorweight. Data were presented as mean � SD. � , P < 0.05; �� , P < 0.01; �� , P < 0.001, Student t test.


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Figure 4.

lncRNA-TTN-AS1 regulates cell metastasis by the EMT signaling pathway. A, Migrating ability of indicated ESCC cell lines was measured (original magnification,200�). B and C, The protein (B) and mRNA (C) levels of epithelial and mesenchymal markers in indicated ESCC cell lines. D, In vivo images of tumor metastasisof mice in each group over time after tail vein injection with indicated ESCC cell lines. Fluorescence intensities of liver (E) and lung (F) in the mice from D.Quantification of metastatic nodes in liver (G) and lung (H) in the different groups. Data were presented as mean � SD. � , P < 0.05; ��, P < 0.01; �� , P < 0.001,Student t test.






imaging indicated that the different clones labeled with GFPmainly distributed in the livers and lungs of nude mice (Fig.4D), overexpression of lncRNA-TTN-AS1 augmented the fluores-cent intensities of liver and lung,whichwere attenuated by ectopicexpression of miR-133b (Fig. 4E and F). Similarly, metastatictumor cells in liver and lung were significantly increased withectopic expression of lncRNA-TTN-AS1, whereas miR-133b abro-gated the increase (Fig. 4G and H; Supplementary Fig. S7F andS7G). All above data verify that lncRNA-TTN-AS1 promotes ESCCmetastasis.

lncRNA-TTN-AS1 enhances FSCN1 expression via its spongeactivity and interaction with HuR

miR-133b targets andmodulates FSCN1 (actin-binding protein,Fascin homolog1) expression that is associated with ESCC cellmetastasis (9). Because lncRNA-TTN-AS1 harbors an identicalmiR-133b–binding site with FSCN1, we questioned whetherlncRNA-TTN-AS1 may regulate FSCN1 by miR-133b and furtherenhances the EMT signaling pathway in ESCC cells. Consistently,overexpression of lncRNA-TTN-AS1 augmented FSCN1 levelwhile ectopic expression ofmiR-133b abrogated the increase. (Fig.5A and B). To further test whether the above effect correlatedto modulation of the FSCN1 30UTR, a luciferase plasmid (pmir-GLO or pmirGLO-FSCN1) was transfected into TE13 cells. Over-expression of lncRNA-TTN-AS1 increased the luciferase intensityof pmirGLO-FSCN1. Ectopic expression of miR-133b overcamethis upregulation (Fig. 5C). In addition, lncRNA-TTN-AS1 tran-script levels were significantly positively correlated with FSCN1mRNA levels in ESCC specimens (Spearman correlation ¼0.9231, P < 0.001) and cell lines (Spearman correlation ¼0.9167, P < 0.001; Supplementary Fig. S8A and S8B). Collectively,these data indicated that the underlying mechanism by whichlncRNA-TTN-AS1 induced the EMT cascade is also associated withpromotion of FSCN1 expression via its sponge activity.

Subcellular location of lncRNAs determines its underlyingmechanism. Cytoplasmic lncRNAs are well known for regulatinggene transcription through interaction with RNA-binding pro-teins (RBP; ref. 11). BecausemiR-133b targets andmodulates HuRmRNA, on the contrary, HuR also repressesmiR-133b release fromlinc-MD1 (12). Thus, we inferred that lncRNA-TTN-AS1 mayalso promote HuR via its sponge activity, which further inducesFSCN1 mRNA expression and stability. As expected, first, ectopicexpression of miR-133b reduced HuR levels (Supplementary Fig.S8C), conversely, depletion of HuR increased miR-133b levels inTE13 cells (Supplementary Fig. S8D). Second, overexpression oflncRNA-TTN-AS1 upregulated HuR levels, which was abolishedby ectopic expression of miR-133b (Fig. 5D). Moreover, ectopicexpression ofmiR-133b abolished the increase in luciferase inten-sity of pmirGLO-HuR induced by overexpression of lncRNA-TTN-AS1 (Supplementary Fig. S8I), indicating that lncRNA-TTN-AS1enhances HuR expression via sponging miRNA-133b. Third, HuRdirectly interacted with both lncRNA-TTN-AS1 and FSCN1mRNAin RIP assays (Fig. 5E). Furthermore, the 50-end (768–518 nt) oflncRNA-TTN-AS1 is indispensable for the interaction betweenlncRNA-TTN-AS1 and HuR (Fig. 5F). Fourth, lncRNA-TTN-AS1overexpression increased the level of HuR protein in the cyto-plasm (Supplementary Fig. S8E), suggesting that lncRNA-TTN-AS1may induce HuR translocation to stabilize FSCN1mRNA. Asexpected, overexpression of lncRNA-TTN-AS1 elongated the half-life of FSCN1 mRNA, which was overcome upon depletion ofHuR (Fig. 5G). In addition, miR-133b inhibited the increase in

FSCN1mRNA stability upon overexpression of lncRNA-TTN-AS1(Fig. 5H). Taken together, on one hand, lncRNA-TTN-AS1 inducesHuR expression via competitive binding of miR-133b, whichfurther enhances FSCN1mRNA stability through binding ofHuR.On the other hand, lncRNA-TTN-AS1 also stabilizes FSCN1mRNA via its sponge activity.

Given that FSCN1 is a downstream target of b-catenin (13) andb-catenin interacts with HuR (14), we first examined the effect ofmodulating lncRNA-TTN-AS1 on the HuR and b-catenin levels inTE-13 cells. Ectopic expression of lncRNA-TTN-AS1 induced HuRexpression (Fig. 5D) and a concomitant upregulation and nuclearaccumulation of b-catenin (Supplementary Figs. S8F and S5I).Second, silencing ofHuR decreased b-catenin expression (Fig. 5J),and knockdown of b-catenin reduced FSCN1 (Fig. 5K), suggestingthe existence of a concerted lncRNA-TTN-AS1 >HuR > b-catenin >FSCN1 axis. Notably, HuR deficiency overcame the effect oflncRNA-TTN-AS1 in promoting FSCN1 expression (Supplemen-tary Fig. S8G), cell proliferation (Fig. 5L), and migration (Sup-plementary Fig. S8H) in ESCC cells, suggesting HuR also pre-dominately modulates the function of lncRNA-TTN-AS1. In sum-mary, lncRNA-TTN-AS1 enhances HuR expression via spongingmiR-133b, and upregulation of HuR further decreases miR-133blevel and increases b-catenin expression, resulting in upregulationof FSCN1.

lncRNA-TTN-AS1/miR-133b/FSCN1 as a biomarker panel inESCC

To investigate the association between lncRNA-TTN-AS1/miR-133b/FSCN1 levels and ESCC progression, we measuredthe expression levels of lncRNA-TTN-AS1/miR-133b/FSCN1 incohorts 1 and 2 by qRT-PCR and ISH assays, respectively. Theresults showed that lncRNA-TTN-AS1 and FSCN1 were mainlyexpressed in the ESCC tissues compared with adjacent normaltissues (Supplementary Fig. S3A and S8J, Fig. 6A and 6C), whereasmiR-133b expression was more abundant in the normal tissues(Fig. 6B; Supplementary Fig. S3B).

In cohort 1, lncRNA-TTN-AS1-high group was notably corre-lated with high advanced TNM stage (N stage, P ¼ 0.033) andclinical stage (P¼ 0.013; Supplementary Table S4). The data werealso examined by analysis of samples from cohort 2 (Supple-mentary Table S7). In contrast, in cohort 1,miR-133b-low expres-sion was significantly associated with pathologic grade (P ¼0.075), high advanced TNM stage (N stage, P ¼ 0.033), andclinical stage (P¼ 0.001; Supplementary Table S5). These correla-tions were also validated by analysis of samples from cohort 2(Supplementary Table S8). There was also significant associationbetween FSCN1-high expression and pathologic grade (P ¼0.004), TNM stage (T stage, P ¼ 0.002), N stage (P ¼ 0.000),and clinical stage (P ¼ 0.004; Supplementary Table S6) in cohort1. The data were also validated by analysis of specimens fromcohort 2 (Supplementary Table S9). In addition, Kaplan–Meierand log-rank test analyses verified that patients with high expres-sion of lncRNA-TTN-AS1/FSCN1 or low expression of miR-133bwere positively correlated with a reduced overall survival (OS; P <0.001; Fig. 6D–F). Moreover, the OS of ESCC patients withlncRNA-TTN-AS1-high/miR-133b-low was shorter than patientsin the other three groups (Fig. 6G). Similarly, the lncRNA-TTN-AS1-high/FSCN1-high group has a significantly lower survivalrate compared with the other groups (Fig. 6H). In addition,multivariate analysis indicated that stage (HR, 2.39; 95% CI,1.14–4.96; P ¼ 0.001), lncRNA-TTN-AS1 expression (HR, 2.73;


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Figure 5.

lncRNA-TTN-AS1 increases stability of FSCN1mRNA. ThemRNA (A) and protein (B) levels of FSCN1 in indicated ESCC cell lines. C, Luciferase activity in the indicatedESCC cell lines transfected with luciferase reporters containing FSCN1 30UTR or a mutant disrupting the miR-133b binding site. D, Protein level of HuR inindicated ESCC cell lines. E, Relative enrichment of lncRNA-TTN-AS1 and FSCN1 in RIP using anti-HuR antibody on TE-13 cells. The fold enrichment of lncRNA-TTN-AS1and FSCN1 was normalized to nonspecific IgG as a negative control. F, Biotinylated RNAs corresponding to different fragments of lncRNA-TTN-AS1 or itsantisense sequence (dotted line) were incubated with TE-13 cell lysates and precipitated with streptavidin beads. The biotinylated RNA–associated proteins wereprobed by Western blot analysis. G and H, The stability of FSCN1 and b-actin mRNA was measured by qRT-PCR relative to time 0 after blocking new RNAsynthesis with a-amanitin (50 mmol/L) in indicated TE-13 cell clones or KYSE-410 cell clones and normalized to 18S rRNA (a product of RNA polymerase I that isunchanged by a-amanitin). I, The subcellular levels of b-catenin in KYSE-410 cells after overexpression of lncRNA-TTN-AS1. The protein level of b-catenin (J) andFSCN1 (K) in indicated ESCC cells. L, The cell viability was detected in indicated KYSE-410 cells. Data were presented as mean � SD. � , P < 0.05; �� , P < 0.01;�� , P < 0.001, Student t test.






Figure 6.

The expression profiles of lncRNA-TTN-AS1, miR-133b, and FSCN1 in 80 ESCC patients. Representative expression images of lncRNA-TTN-AS1 (A), miR-133b (B),and FSCN1 (C) expression from ESCC tissues and adjacent normal tissues by ISH assays. Kaplan–Meier survival analysis of the overall survival in ESCC caseswith high versus low lncRNA-TTN-AS1 expression (D), miR-133b expression (E), and FSCN1 expression (F). Kaplan–Meier survival analysis of the overallsurvival in ESCC cases with different lncRNA-TTN-AS1/miR-133b (G) and lncRNA-TTN-AS1/FSCN1 expression pattern (H; P < 0.01). I, A schematic model oflncRNA-TTN-AS1 function during the metastasis cascade.


Lin et al.




95% CI, 1.27–4.58; P ¼ 0.004), miR-133b expression (HR, 2.13;95%CI, 1.36–5.78; P¼ 0.023), and FSCN1 expression (HR, 3.68;95% CI, 1.35–7.02; P ¼0.000) were independent prognosticfactors forOS in ESCCpatients (Supplementary Table S10). Thesedata suggested that combination of lncRNA-TTN-AS1 andmiR-133b or FSCN1 could separate patients into distinct prog-nostic groups (P < 0.01).

DiscussionThe key finding of this study was the discovery of a novel

lncRNA-TTN-AS1 that plays a vital role in ESCC progression andmetastasis. We discovered that lncRNA-TTN-AS1 enhances Snail1and FSCN1 levels by competitively binding tomiR-133b, resultingin the promotion of EMT cascades. Meanwhile, lncRNA-TTN-AS1also induces HuR via its sponge activity, which further activatesthe FSCN1/b-catenin signaling pathway (Fig. 6l). In addition,lncRNA-TTN-AS1/miR-133b/FSCN1 is a potential prognostic bio-marker panel in ESCC carcinogenesis.

The functions of lncRNAs are closely related to their subcellularlocalization. Nuclear lncRNAs mainly affect chromatin structureand gene transcription (15, 16). Cytosolic lncRNAs modulateprotein subcellular localization (17) and mRNA translation (18,19).Herein, lncRNA-TTN-AS1has been shown to targetmiR-133busing multiple bioinformatics platforms. Intriguingly, ectopicexpression of miR-133b decreased the luciferase intensities of thelncRNA-TTN-AS1WT reporter. However, there was no significantdifference in lncRNA-TTN-AS1 expression upon overexpression ofmiR-133b. Moreover, endogenous lncRNA-TTN-AS1 and miR-133b were pulled down by anti-Ago2. Taken together, all thesedata suggest that miR-133b recognizes and binds with lncRNA-TTN-AS1without triggering degradation of lncRNA-TTN-AS1. Thereciprocal modulation between miRNAs and lncRNAs is stillelusive miR-21 and lncRNA-GAS5 suppress expression of eachother in an Ago2-dependent manner (20), whereas miR-200 onlybind to lncRNA-ATB via the RISC complex but does not affect theexpression of lncRNA-ATB (21). Further studies are still requiredto fully understand the miRNA regulatory network.

The miR-133 family has tumor-suppressive genes (22, 23) thatare involved inESCCprogression (9). BecausemiR-133bmediatedSnail1 repression was shown to improve cardiac reprogramming(10), thus, we reasoned that miR-133b may regulate Snail1, andthen induce an invasion cascade in ESCC. Snail1 as an EMTtranscription factor coordinates the repression of epithelial phe-notype and the induction of mesenchymal phenotype (24, 25).lncRNA-TTN-AS1 abolishes the suppression of Snail1 mediatedby miR-133b, and the upregulation of Snail1 contributes to therepression of epithelial markers E-cadherin and ZO-1 and acti-vation of mesenchymal markers N-cadherin and vimentin, pro-moting theEMTcascade.Meanwhile,miR-133bbinds and inhibitsFSCN1 expression (9), which is significantly correlated with poor

prognosis in ESCC (26). Therefore, we assumed that lncRNA-TTN-AS1 could modulate FSCN1mRNA level by competitively spong-ing miR-133b, thereby enhancing ESCC metastasis. As expected,lncRNA-TTN-AS1 increased FSCN1 expression in ESCC cells,which was reversed by miR-133b. In addition, lncRNA-TTN-AS1facilitated HuR expression via sponging miR-133b, the upregula-tion of HuR further decreased miR-133b level and enhancedb-catenin expression, which induced FSCN1 mRNA level andstability. Notably, the combination between lncRNA-TTN-AS1and miR-133b/FSCN1 could be a potential prognostic biomarkerpanel of ESCC. All the above data suggested that the lncRNA–miR-133b–FSCN1 axis plays a critical role in the metastasis-invasioncascade in ESCC progression.

Taken together, our research revealed that lncRNA-TTN-AS1promotes ESCC cell proliferation and invasion metastasis, whichinduces competitive binding to miR-133b, resulting in upregula-tion of Snail1, HuR, and FSCN1. In addition, HuR-induced bylncRNA-TTN-AS1 increases b-catenin expression, enhancingFSCN1 mRNA expression and stability by interaction with HuR.The pleiotropic effect of lncRNA-TTN-AS1 on ESCC progressionhas provided important new insights into our comprehension ofESCC carcinogenesis.

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

Authors' ContributionsConception and design: C. Lin, E. NiceDevelopment of methodology: C. LinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Lin, S. Zhang, M. Li, C. Liu, J. Hao, W. QiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Lin, Ying Wang, Yuanshu Wang, L. Yu, L. HuWriting, review, and/or revision of the manuscript: C. Lin, S. Zhang, C. Guo,E. ZhangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Xu

AcknowledgmentsThe authors are very grateful to Nanjing General Hospital for providing the

ESCC tissues and Prof. Yifeng Zhou (Suzhou University), who provided theESCC cell lines; Project Programof State Key Laboratory of NaturalMedicines inChina (no. SKLNMBZ201403); National Science and Technology Major Pro-jects of New Drugs in China (2012ZX09103301-004 and 2014ZX09508007);and The Priority Academic Program Development of Jiangsu Higher EducationInstitutions (PAPD). This work was supported by the National Natural ScienceFoundation of China (30873073).

The costs of publication of this articlewere defrayed inpart 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 29, 2017; revised October 4, 2017; accepted October 30, 2017;published OnlineFirst November 3, 2017.

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2018;24:486-498. Published OnlineFirst November 3, 2017.Clin Cancer Res Chenyu Lin, Shengnan Zhang, Ying Wang, et al.

MetastasisEsophageal Squamous Cell Carcinoma Progression and inTTN-AS1Functional Role of a Novel Long Noncoding RNA

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Functional Role of a Novel Long Noncoding RNA in ... · Esophageal carcinoma is the sixth leading cause of tumor-related mortality worldwide (1). There are two main esophageal carcinoma