Xu et al. Cell Biosci (2018) 8:61 https://doi.org/10.1186/s13578-018-0260-0 Cell & Bioscience RESEARCH Open Access TUG1 confers cisplatin resistance in esophageal squamous cell carcinoma by epigenetically suppressing PDCD4 expression via EZH2 Caihui Xu1, Yinmou Guo1*, Haiyan Liu1, Gongbin Chen1, Yanju Yan1 and Teng Liu2 Abstract Background: Increasing evidence has suggested the involvement of long non-coding RNA taurine upregu- lated gene 1 (TUG1) in chemoresistance of cancer treatment. However, its function and molecular mechanisms in esophageal squamous cell carcinoma (ESCC) chemoresistance are still not well elucidated. In the present study, we investigate the functional role of TUG1 in cisplatin (DDP) resistance of ESCC and discover the underlying molecular mechanism. Results: Our study revealed that TUG1 was up-regulated in DDP-resistant ESCC tissues and cells. High TUG1 expres- sion was correlated with poor prognosis of ESCC patients. TUG1 knockdown improved the sensitivity of ECA109/ DDP and EC9706/DDP cells to DDP. Moreover, TUG1 could epigenetically suppress PDCD4 expression via recruit- ing enhancer of zeste homolog 2. PDCD4 overexpression could mimic the functional role of down-regulated TUG1 in DDP resistance. PDCD4 knockdown counteracted the inductive efect of TUG1 inhibition on DDP sensitivity of ECA109/DDP and EC9706/DDP cells. Furthermore, TUG1 knockdown facilitated DDP sensitivity of DDP-resistant ESCC cells in vivo. Conclusion: TUG1 knockdown overcame DDP resistance of ESCC by epigenetically silencing PDCD4, providing a novel therapeutic target for ESCC. Keywords: Esophageal squamous cell carcinoma, Cisplatin, Taurine upregulated gene 1, Enhancer of zeste homolog 2, PDCD4 Background achieve successful treatment for ESCC [5, 6]. Terefore, Esophageal squamous cell carcinoma (ESCC) is one it is urgent to elucidate the mechanism underlying chem- of the most frequent gastrointestinal malignancies in oresistance in ESCC and develop novel therapeutic strat- human, a sixth most common cause of cancer-related egies to improve ESCC prognosis. death worldwide [1, 2]. Despite the development of Long noncoding RNAs (lncRNAs) represent a class of surgery combined with neoadjuvant radiation and/or endogenous non-protein-coding RNAs longer than 200 chemotherapy, the majority of patients with ESCC were nucleotides [7]. Emerging evidence suggests the involve- diagnosed frequently at the advanced stage and had poor ment of lncRNAs in normal development as well as prognosis [3, 4]. Chemoresistance frequently occurs tumorigenesis [8]. Dysregulated lncRNAs could act as during chemotherapy, which remains a major barrier to oncogenic molecules and tumor suppressors in malignant tumors, closely associated with tumorigenesis, metasta- *Correspondence: [email protected] sis, diagnosis or prognosis [9]. Moreover, accumulating 1 Department of Oncology, Shangqiu First People’s Hospital, No. 292 documents revealed that abnormal lncRNAs were related Kaixuan South Road, Shangqiu 476100, China Full list of author information is available at the end of the article to chemotherapy resistance of cancers [10–12]. LncRNA © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. Cell Biosci (2018) 8:61 Page 2 of 11 taurine-upregulated gene 1 (TUG1), located on chromo- (si-PDCD4) and their scramble negative control (si-con) some 22q12.2, was originally identifed as a transcript were chemically synthesized by Genepharma (Shanghai, up-regulated by taurine [13]. Recently, increasing evi- China). All cell transfections were performed using the dence had suggested that aberrant TUG1 expression was Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). associated with non-small cell lung cancer, hepatocellu- lar carcinoma, and ESCC [14–16]. Although a previous Quantitative real‑time PCR (qRT‑PCR) study reported that high TUG1 expression was signif- Total RNA was isolated from ESCC tissues and cells cantly correlated with chemotherapy resistance in ESCC, using Trizol reagent (Invitrogen) and then reversely tran- the function and mechanism of TUG1 in cisplatin (DDP) scribed into cDNA using PrimeScript RT Reagent Kit resistance of ESCC remains uncertain. (TaKaRa, Dalian, China). TUG1 and PDCD4 expression In this study, we aimed to investigate the expression levels were detected by quantitative real-time PCR with and functional role of TUG1 in ESCC DDP resistance as SYBR Green Master Mix (TOYOBO, Osaka, Japan) using well as its underlying molecular mechanism. Our study an Applied Biosystems 7500 Real-Time PCR Systems found that TUG1 expression was increased in ESCC tis- (Applied Biosystems, Foster City, CA, USA). Te primes sues and cell lines, especially in DDP-resistant tissues were as follows: TUG1 forward, 5′-TAG CAG TTC CCC and cells. Functionally, TUG1 knockdown improved the AAT CCT TG-3′, TUG1 reverse, 5′-CAC AAA TTC CCA sensitivity of DDP-resistant ESCC cells to DDP. Mechani- TCA TTC CC-3′; PDCD4 forward, 5′-GGC CTC CAA cally, TUG1 improved the sensitivity of ESCC cells to GGA GTA AGA CC-3′, PDCD4 reverse, 5′-AGG GGT CTA DDP through epigenetically suppressing PDCD4 expres- CAT GGC AAC TG-3′. Data were analyzed using the com- sion through recruiting enhancer of zeste homolog 2 parative Ct method (2−ΔΔCt) with GAPDH as an internal (EZH2). Our study revealed a novel epigenetical regu- control. latory mechanism between TUG1 and PDCD4 which could overcome DDP resistance in ESCC. Drug sensitivity assay Te cell viability of ECA109/DDP and EC9706/DDP cells Methods and their parental cells was measured by 3-(4,5-dimethyl- Sample collection and cell culture 2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) Te paired tumor tissues and adjacent normal tissues (Sigma, St. Louis, Missouri, USA) assay. DDP sensitiv- (n = 42) were collected from ESCC patients who under- ity was determined using the IC50 value (half maximal went surgery resection at the Shangqiu frst People’s Hos- inhibitory concentration). pital. Tis study was approved by the Ethics Committee of Shangqiu frst People’s Hospital and informed consents Flow cytometric analysis were signed by all patients. Te normalized RNA-seq Cell apoptosis was evaluated using Annexin V-FITC/ data of Esophageal Carcinoma (ESCA) were downloaded PI Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, from the TCGA data portal website (https ://cance rgeno China) as described previously [18]. Briefy, ECA109/ me.nih.gov/). DDP and EC9706/DDP cells with diferent transfection Human immortalized esophageal epithelial cell line were treated with 20 μM DDP for 48 h, followed by dou- HET-1A and human ESCC cell lines (ECA109 and ble stained with Annexin V-FITC and PI under a dark EC9706) were purchased from ATCC (Manassas, VA, condition. Cell apoptotic rates were evaluated by FACSan USA). All cells were cultured in PRMI-1640 medium fow cytometry (BD Biosciences, San Jose, CA, USA). (Gibco, Rockville, MD, USA) supplemented with 10% Subcellular fraction assays FBS (Gibco) at 37 °C with 5% CO2. DDP-resistant vari- ants (ECA109/DDP and EC9706/DDP) of ECA109 and Te separation of the nuclear and cytosolic fractions of EC9706 cells were established using a repetitive pulsatile ECA109 cells was performed using the PARIS Kit (Life treatment with constant concentrations of cisplatin [17]. Technologies, Carlsbad, CA, USA) following the manu- Te degree of chemotherapy resistance of DDP-resistant facturer’s instructions. variants was evaluated before transfections. RNA pull‑down assays Cell transfection TUG1 and anti-sense-TUG1 was transcribed with Tran- Empty pcDNA3.1 vector (Vector) was obtained from scriptAid T7 High Yield Transcription Kit (Termo Genepharma (Shanghai, China). TUG1 or PDCD4 Fisher Scientifc) and then labeled with Termo Scientifc overexpressing vector pcDNA3.1-TUG1 or pcDNA3.1- Pierce RNA 3′ Desthiobiotinylation Kit (Termo Fisher PDCD4 (TUG1 or PDCD4), small interfering RNAs Scientifc). Pierce Magnetic RNA-Protein Pull down Kit against TUG1 (si-TUG1 #1 or si-TUG1 #2) or PDCD4 (Termo Fisher Scientifc) was used to perform RNA Xu et al. Cell Biosci (2018) 8:61 Page 3 of 11 pull down assay. Briefy, labeled RNAs were bound with stable lentivirus-transfected ECA109/DDP cell line. 7 Streptavidin Magnetic Beads and then incubated with Ten, ECA109/DDP cells (1.0 × 10 ) stably infected with ECA109/DDP cell protein lysates. Ten the RNA-binding sh-TUG1 or sh-con were subcutaneously injected into proteins were eluted for the further western blot analysis. the tail veins of BALB/c-nude mice (4 weeks old) from the Shanghai Experimental Animal Center of the Chi- RNA immunoprecipitation (RIP) assays nese Academy of Sciences (Shanghai, China). One week RIP experiments were performed using Magna RIP™ later, mice were intraperitoneally injected with 6 mg/ RNA-Binding Protein Immunoprecipitation Kit (Mil- kg DDP or same volume of PBS every week according lipore, Bedford, MA, USA) according to the manufac- to indicated groups (n = 5 each group): sh-con + PBS, turer’s protocol. EZH2 and IgG antibodies