TUG1 confers cisplatin resistance in esophageal squamous cell carcinoma by epigenetically suppressing PDCD4 expression via EZH2
Increasing evidence has suggested the involvement of long non-coding RNA taurine upregulated 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.
Our study revealed that TUG1 was up-regulated in DDP-resistant ESCC tissues and cells. High TUG1 expression 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 recruiting enhancer of zeste homolog 2. PDCD4 overexpression could mimic the functional role of down-regulated TUG1 in DDP resistance. PDCD4 knockdown counteracted the inductive effect 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.
TUG1 knockdown overcame DDP resistance of ESCC by epigenetically silencing PDCD4, providing a novel therapeutic target for ESCC.
KeywordsEsophageal squamous cell carcinoma Cisplatin Taurine upregulated gene 1 Enhancer of zeste homolog 2 PDCD4
Esophageal squamous cell carcinoma (ESCC) is one of the most frequent gastrointestinal malignancies in human, a sixth most common cause of cancer-related death worldwide [1, 2]. Despite the development of surgery combined with neoadjuvant radiation and/or chemotherapy, the majority of patients with ESCC were diagnosed frequently at the advanced stage and had poor prognosis [3, 4]. Chemoresistance frequently occurs during chemotherapy, which remains a major barrier to achieve successful treatment for ESCC [5, 6]. Therefore, it is urgent to elucidate the mechanism underlying chemoresistance in ESCC and develop novel therapeutic strategies to improve ESCC prognosis.
Long noncoding RNAs (lncRNAs) represent a class of endogenous non-protein-coding RNAs longer than 200 nucleotides . Emerging evidence suggests the involvement of lncRNAs in normal development as well as tumorigenesis . Dysregulated lncRNAs could act as oncogenic molecules and tumor suppressors in malignant tumors, closely associated with tumorigenesis, metastasis, diagnosis or prognosis . Moreover, accumulating documents revealed that abnormal lncRNAs were related to chemotherapy resistance of cancers [10, 11, 12]. LncRNA taurine-upregulated gene 1 (TUG1), located on chromosome 22q12.2, was originally identified as a transcript up-regulated by taurine . Recently, increasing evidence had suggested that aberrant TUG1 expression was associated with non-small cell lung cancer, hepatocellular carcinoma, and ESCC [14, 15, 16]. Although a previous study reported that high TUG1 expression was significantly correlated with chemotherapy resistance in ESCC, the function and mechanism of TUG1 in cisplatin (DDP) resistance of ESCC remains uncertain.
In this study, we aimed to investigate the expression and functional role of TUG1 in ESCC DDP resistance as well as its underlying molecular mechanism. Our study found that TUG1 expression was increased in ESCC tissues and cell lines, especially in DDP-resistant tissues and cells. Functionally, TUG1 knockdown improved the sensitivity of DDP-resistant ESCC cells to DDP. Mechanically, TUG1 improved the sensitivity of ESCC cells to DDP through epigenetically suppressing PDCD4 expression through recruiting enhancer of zeste homolog 2 (EZH2). Our study revealed a novel epigenetical regulatory mechanism between TUG1 and PDCD4 which could overcome DDP resistance in ESCC.
Sample collection and cell culture
The paired tumor tissues and adjacent normal tissues (n = 42) were collected from ESCC patients who underwent surgery resection at the Shangqiu first People’s Hospital. This study was approved by the Ethics Committee of Shangqiu first People’s Hospital and informed consents were signed by all patients. The normalized RNA-seq data of Esophageal Carcinoma (ESCA) were downloaded from the TCGA data portal website (https://cancergenome.nih.gov/).
Human immortalized esophageal epithelial cell line HET-1A and human ESCC cell lines (ECA109 and EC9706) were purchased from ATCC (Manassas, VA, USA). All cells were cultured in PRMI-1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% FBS (Gibco) at 37 °C with 5% CO2. DDP-resistant variants (ECA109/DDP and EC9706/DDP) of ECA109 and EC9706 cells were established using a repetitive pulsatile treatment with constant concentrations of cisplatin . The degree of chemotherapy resistance of DDP-resistant variants was evaluated before transfections.
Empty pcDNA3.1 vector (Vector) was obtained from Genepharma (Shanghai, China). TUG1 or PDCD4 overexpressing vector pcDNA3.1-TUG1 or pcDNA3.1-PDCD4 (TUG1 or PDCD4), small interfering RNAs against TUG1 (si-TUG1 #1 or si-TUG1 #2) or PDCD4 (si-PDCD4) and their scramble negative control (si-con) were chemically synthesized by Genepharma (Shanghai, China). All cell transfections were performed using the Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from ESCC tissues and cells using Trizol reagent (Invitrogen) and then reversely transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). TUG1 and PDCD4 expression levels were detected by quantitative real-time PCR with SYBR Green Master Mix (TOYOBO, Osaka, Japan) using an Applied Biosystems 7500 Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA). The primes were as follows: TUG1 forward, 5′-TAGCAGTTCCCCAATCCTTG-3′, TUG1 reverse, 5′-CACAAATTCCCATCATTC CC-3′; PDCD4 forward, 5′-GGCCTCCAAGGAGTAAGACC-3′, PDCD4 reverse, 5′-AGGGGTCTACATGGCAACTG-3′. Data were analyzed using the comparative Ct method (2−ΔΔCt) with GAPDH as an internal control.
Drug sensitivity assay
The cell viability of ECA109/DDP and EC9706/DDP cells and their parental cells was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, Missouri, USA) assay. DDP sensitivity was determined using the IC50 value (half maximal inhibitory concentration).
Flow cytometric analysis
Cell apoptosis was evaluated using Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN Biotech, Nanjing, China) as described previously . Briefly, ECA109/DDP and EC9706/DDP cells with different transfection were treated with 20 μM DDP for 48 h, followed by double stained with Annexin V-FITC and PI under a dark condition. Cell apoptotic rates were evaluated by FACSan flow cytometry (BD Biosciences, San Jose, CA, USA).
Subcellular fraction assays
The separation of the nuclear and cytosolic fractions of ECA109 cells was performed using the PARIS Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions.
RNA pull-down assays
TUG1 and anti-sense-TUG1 was transcribed with TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) and then labeled with Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit (Thermo Fisher Scientific). Pierce Magnetic RNA-Protein Pull down Kit (Thermo Fisher Scientific) was used to perform RNA pull down assay. Briefly, labeled RNAs were bound with Streptavidin Magnetic Beads and then incubated with ECA109/DDP cell protein lysates. Then the RNA-binding proteins were eluted for the further western blot analysis.
RNA immunoprecipitation (RIP) assays
RIP experiments were performed using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s protocol. EZH2 and IgG antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The co-precipitated RNAs were purified and analyzed by qRT-PCR analysis.
Chromatin immunoprecipitation (ChIP) assays
Chromatin immunoprecipitation assay was performed to confirm the interaction between TUG1 and PDCD4 gene using EZ-ChIP kit (Millipore). The chromatins were immunoprecipitated with antibodies against EZH2 (Cell Signaling Technology), H3K27me3 (Millipore) or IgG (Millipore). Finally, the immunoprecipitated chromatin was purified and analyzed by qRT-PCR analysis. Primers for PDCD4 promoter region were 5′-GGTCTGGGAAGCTCCGATTT-3′ (forward) and 5′-GCAGTTGGTGGTCATCCTCA-3′ (reverse).
Luciferase reporter assay
PDCD4 promoter sequences were inserted into pGL3-Basic luciferase plasmid (Promega, Madison, WI, USA) to generate PDCD4 promoter reporter vector. Then, PDCD4 promoter reporter was transfected into ECA109/DDP cells using Lipofectamine 2000 (Invitrogen) along with phRL-TK vector (Promega) and (Vector or TUG1) or (si-con or si-TUG1). Luciferase Reporter assay system (Promega) was performed to detect luciferase activity in ECA109/DDP cells 48 h post-transfection.
Western blot analysis
Western blotting was performed according to our previously reported protocol . The primary antibodies anti-EZH2, anti-PDCD4 and anti-GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).
The animal experiment was performed according to the national standard of the care and use of laboratory animals and got the approval of the Ethics Committee of Shangqiu first People’s Hospital. ECA109/DDP cells were infected with sh-TUG1 or sh-con lentivirus, followed by the sieving using puromycin (Sigma-Aldrich, St. Louis, MO, USA) for nearly 7 days to construct stable lentivirus-transfected ECA109/DDP cell line. Then, ECA109/DDP cells (1.0 × 107) stably infected with sh-TUG1 or sh-con were subcutaneously injected into the tail veins of BALB/c-nude mice (4 weeks old) from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). One week later, mice were intraperitoneally injected with 6 mg/kg DDP or same volume of PBS every week according to indicated groups (n = 5 each group): sh-con + PBS, sh-TUG1 + PBS, sh-con + DDP, sh-TUG1 + DDP. The tumor sizes were measured every week. After 42 days, the mice were killed, and the tumor weights were detected. qRT-PCR and western blot assays were performed to detect TUG1 expression and PDCD4 protein levels.
All data were presented as means ± standard deviation (SD). Student’s t-test and one-way ANOVA were used to calculate the statistic difference using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Differences were considered statistically significant when P value < 0.05.
TUG1 was increased in DDP-resistant ESCC tissues and cells
TUG1 knockdown overcame DDP resistance of ESCC cells
TUG1 epigenetically suppressed PDCD4 expression in ESCC cells
PDCD4 overexpression enhanced DDP sensitivity of ESCC cells
TUG1 knockdown facilitated DDP sensitivity of ESCC cells through increasing PDCD4 expression
TUG1 knockdown enhanced DDP sensitivity in tumors in vivo
Acquiring chemoresistance have restricted treatment outcome for ESCC patients in the clinic. Hence, it is essential to investigate the molecular mechanism underlying chemoresistance and identify novel targets for chemoresistance therapy. In this study, we found that the expression level of TUG1 was significantly elevated in DDP-resistant ESCC tissues and cells. Moreover, TUG1 knockdown re-sensitized ECA109/DDP and EC9706/DDP cells to DDP by promoting DDP-induced apoptosis. More importantly, TUG1 conferred DPP resistance to ESCC cells via epigenetically silencing PDCD4 via EZH2. Therefore, TUG1 may be a promising therapeutic target for DDP resistance in ESCC.
Elucidating the molecular mechanism underlying chemoresistance could contribute to develop reasonable and effective therapies to overcome chemoresistance. Our results demonstrated that TUG1 expression level was elevated in DDP-resistant ESCC tissues and cells, and down-regulation of TUG1 re-sensitized ECA109/DDP and EC9706/DDP cells to DDP. Apart from our findings, dysregulated TUG1 has been reported to be implicated with chemoresistance in other cancers. For example, TUG1 was overexpressed in small cell lung cancer, and TUG1 down-regulation sensitized lung cancer cells to chemotherapeutic drugs (DDP, Adriamycin and Etoposide) by epigenetically suppressing LIM-kinase 2b (LIMK2b) expression through EZH2 . Moreover, TUG1 knockdown re-sensitized MTX-resistant colorectal cell lines to MTX through acting as a competitive endogenous RNA (ceRNA) to sponge miR-186 and release the miRNA target CPEB2 . On the contrary, TUG1 expression was down-regulated in triple negative breast cancer, and overexpression of TUG1 enhance DDP sensitivity in MDA-MB-231 and BT549 cells by sponging miR-197 . All these findings suggested that the TUG1 could be used as a promising therapeutic target for chemoresistance in cancers.
The precise mechanism by which TUG1 up-regulation contributed to DDP resistance in ESCC was unclear. Hence, the functional mechanism of TUG1 was further investigated in the present study. Previous studies found that about 20% of lncRNAs can bind to polycomb repressive complex 2 (PRC2), which subsequently induced the silence of targeted genes through harboring methyltransferase activity . Moreover, TUG1 has been proved to regulate genes expression by binding with EZH2 in human non-small cell lung cancer, gastric cancer and hepatocellular carcinoma [14, 26, 27]. EZH2, a vital catalytic subunit of PRC2, is a histone methyltransferase that epigenetically represses gene expression by promoting histone H3 lysine 27 trimethylation (H3 K27me3) [28, 29]. PDCD4, a tumor suppressor, was recently demonstrated to be negatively regulated by CASC15, via recruiting EZH2 and subsequently changing H3 K27me3 level in melanoma . Therefore, we further investigated whether TUG1 could regulate PDCD4 expression by recruiting EZH2. Our western blot assays indicated that TUG1 or EZH2 knockdown elevated PDCD4 protein levels. Moreover, RNA pull-down and RIP assays further validated that TUG1 could bind to EZH2. ChIP and luciferase reporter assays further proved that TUG1 knockdown enhanced the promoter activity of PDCD4 by attenuating the recruiting of EZH2 on PDCD4 promoter region. These data demonstrated that TUG1 epigenetically silencing PDCD4 via recruiting EZH2 in ECA109/DDP cells. PDCD4 has been identified as a tumor suppressor in multiple cancers [31, 32]. Moreover, PDCD4 could improve the sensitivity of cancer cells to chemotherapy drugs such as docetaxel and cisplatin [33, 34]. Particularly, overexpression of PDCD4 induced apoptosis and enhanced chemosensitivity to cisplatin in ESCC . Consistently, our data also revealed that PDCD4 overexpression could overcome DDP resistance in ECA109/DDP and EC9706/DDP cells. Furthermore, PDCD4 inhibition reversed the inductive effect of TUG1 knockdown on the sensitivity of ECA109/DDP and EC9706/DDP to DDP. All these data demonstrated that TUG1 inhibition sensitized DDP-resistant ESCC cells to DDP through epigenetically silencing PDCD4 in ESCC.
In conclusion, our study demonstrated that TUG1 knockdown enhanced DDP sensitivity of ESCC cells. Importantly, the enhancive effect of TUG1 inhibition on DDP sensitivity might be mediated by PDCD4 through an epigenetic mechanism in ESCC cells, providing a promising therapeutic strategy to overcome DDP resistance in ESCC.
CX and HL designed the experiments. GC, YY and TL performed the experiments and acquired the data. CX and YG prepared the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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Ethics approval and consent to participate
This study was approved by the Ethics Committee of Shangqiu first People’s Hospital and informed consents were signed by all patients.
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