Published OnlineFirst July 1, 2016; DOI: 10.1158/1535-7163.MCT-16-0008 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Long Noncoding RNA MALAT1 Promotes Aggressive Pancreatic Cancer Proliferation and Metastasis via the Stimulation of Autophagy Le Li1, Hua Chen1, Yue Gao2, Yong-Wei Wang1, Guang-Quan Zhang1, Shang-Ha Pan1, Liang Ji1, Rui Kong1, Gang Wang1, Yue-Hui Jia3, Xue-Wei Bai1, and Bei Sun1 Abstract Recently, pancreatic ductal adenocarcinoma (PDAC) has LC3, P62, and LAMP-2. Mechanistically, we found that MALAT1 emerged as one of the most aggressive malignant tumors with interacted with RNA binding protein HuR, and silencing of the worst prognosis. Previous studies have demonstrated that MALAT1 greatly enhanced the posttranscriptional regulation long noncoding RNA metastasis-associated lung adenocarcinoma of TIA-1 and had further effects on inhibiting autophagy. transcript 1 (MALAT1) is increased in pancreatic cancer and is MALAT1 was speculated to regulate tumorigenesis via HuR- identified as a diagnostic biomarker. Nonetheless, the molecular TIA-1–mediated autophagic activation. Hence, we investigated mechanism of elevated MALAT1 levels and tumor aggressiveness the biological properties of MALAT1 in terms of tumor prolif- remains unknown. In this study, MALAT1 was found to be highly eration and metastasis by promoting autophagy in vitro. In expressed in PDAC tissues, and elevated expression was associated brief, these data demonstrate that MALAT1 could facilitate with poorer prognoses. In addition, MALAT1 was positively the advanced progression of tumors in vivo. Our study high- linearly correlated with the expression of LC3B mRNA. Further- lights the new roles of MALAT1 on protumorigenic functioning more, several molecules involved in cellular autophagic flux were and anticancer therapy via activating autophagy in pancreatic modulated following the downregulation of MALAT1, including cancer. Mol Cancer Ther; 15(9); 2232–43. Ó2016 AACR. Introduction 1 (MALAT1), also known as noncoding nuclear-enriched abun- dant transcript 2 (NEAT2), is highly conserved among mammals Pancreatic ductal adenocarcinoma (PDAC) is one of the most and is strongly expressed in the nucleus. MALAT1 was first aggressive malignancies. The overall 5-year survival of PDAC identified as a sign of metastasis in lung cancer (7). Similarly, ranges from 3% to 5%, even with curative tumor resection and early reports demonstrated that MALAT1 was highly expressed in chemotherapy (1). The grave prognosis associated with PDAC is pancreatic cancer hepatocellular carcinoma, prostate cancer, and primarily due to delayed diagnoses and a lack of effective treat- other malignancies (8–10). Few studies have confirmed the strong ment, which emphasizes the importance of identifying PDAC in association between MALAT1 and poor pancreatic cancer prog- earlier stages and establishing better therapeutic targets (2). noses (11). However, the molecular mechanism for its cancer- Long noncoding RNA (lncRNA) is a class of ncRNA with a promoting effect is yet to be defined. length of more than 200 NTs. So far, a large number of lncRNAs Autophagy is an evolutionarily conserved catabolic process and have been recognized by high-throughput transcriptome analysis mechanism for the degradation of cellular proteins, which is (HTTA). They play important regulatory roles in the expression of functional in cell growth regulation and intracellular homeostasis nearby genes, the activation and localization of proteins, and the (12, 13). Autophagy seems complicated and has both protumori- organization of small RNAs (3). LncRNAs are also critical regu- genic and tumor-suppressive roles in pancreatic cancer. It has also lators in tumor-related apoptosis, autophagy, and tumor metas- been described as a moderator of cellular invasion and metastasis tasis (4–6). Metastasis-associated lung adenocarcinoma transcript by impacting cell biological phenotypes (14–16). Increasing evidence suggests that lncRNAs regulate the process of autophagy by means of interacting with RNA-binding proteins (RBP), as well 1Department of Pancreatic and Biliary Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China. as by suppressing the induction of miRNAs (17). However, the 2Department of General Surgery, Cleveland Clinic Foundation, Cleve- existence of this regulatory effect in pancreatic cancer is still under land, Ohio. 3Department of Epidemiology and Biostatistics, School of investigation. Public Health, Qiqihar Medical University, Qiqihar, Heilongjiang, China. In this study, we correlated the clinical significance of MALAT1 Note: Supplementary data for this article are available at Molecular Cancer expression with the level of autophagy in PDAC samples. Then, we Therapeutics Online (http://mct.aacrjournals.org/). explored the roles of MALAT1 in tumor progression and found Corresponding Author: Bei Sun, Department of Pancreatic and Biliary Surgery, that MALAT1 upregulated cellular autophagy in pancreatic cancer The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, cell lines. Furthermore, our study also demonstrated that MALAT1 Nangang District, Harbin 150001, Heilongjiang Province, China. Phone: 8645- directly interacts with HuR and alters the biological process of 1855-55721; Fax: 8645-1536-43849; E-mail: [email protected] autophagy via enhancing the regulation of TIA-1 posttranscrip- doi: 10.1158/1535-7163.MCT-16-0008 tionally. Our study eventually suggested that MALAT1 may accel- Ó2016 American Association for Cancer Research. erate the proliferation and metastasis of cancer cells through the 2232 Mol Cancer Ther; 15(9) September 2016 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst July 1, 2016; DOI: 10.1158/1535-7163.MCT-16-0008 MALAT1 Promotes Cancer Proliferation and Metastasis stimulation of autophagy. MALAT1 functioned as a protumori- The yellow dots that result from merging the red and green genic oncogene in pancreatic cancer in vitro and in vivo. channels indicate autophagosomes, while the red dots that not overlay with green dots are indicative of autolysosomes. The Materials and Methods number of GFP and mRFP dots was determined by manual counting of the fluorescent puncta in five high-power fields Cell culture (40Â, Olympus). Bxpc-3 and Panc-1 and human pancreatic duct epithelial (HPDE) cells were purchased from the ATCC from 2010. All cell lines were authenticated by short tandem repeat (STR) and RNA pull-down assay genotyped upon re-expansions and tested within 6 months of The full-length MALAT1 fragment cleaved by the EcoRI enzyme authentication. Aspc-1, CFPAC, and SW1990 were purchased was excised from the vector pCRII-TOPO. RNA transcript probes from the Type Culture Collection of the Chinese Academy of were synthesized using a MEGAscript T7 kit (Ambion), following Sciences (Shanghai, China) and used within 6 months after the the protocol provided by the manufacturer. A biotinylated RNA cells were ordered. Aspc-1, Bxpc-3, and Panc-1 were cultured in probe synthesized in a 20 mL MEGAscript transcription reaction by RPMI1640 medium (Hyclone), and CFPAC, SW1990, and HPDE adding 1.25 mL 20 mmol/L biotinylated UTP, Biotin-16-UTP were cultured in DMEM (Gibco), all supplemented with 10% FBS (Roche). The synthesized RNA probes were purified using an (Gibco), 1% penicillin, and streptomycin at 37 C with 5% CO2. RNeasy Protect Mini kit (Qiagen). Whole cells were grown to 90% confluence and washed three times with PBS. Then, the cells Patients and specimens were resuspended in CHAPS buffer (10 mmol/L Tris-HCl pH 7.4, This study was approved by the Ethics Committee of the First 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.5% CHAPS, 10% glycerol, Affiliated Hospital of Harbin Medical University. The 52 PDAC 0.1 mmol/L PMSF, 5 mmol/L 2-ME) and incubated 30 minutes specimens used in this experiment were obtained from patients on ice for lysing. The cell lysates were obtained by centrifugation at  who underwent pancreaticoduodenectomy for pancreatic head 10,000 g for 10 minutes at 4 C. The supernatants were collected cancer in the Department of Pancreatic and Biliary Surgery (The and the protein concentration was determined using the Bio-Rad m First Affiliated Hospital of Harbin Medical University, Harbin, protein assay (Bio-Rad). The reaction mixture contained 200 gof m 0 Heilongjiang, China) from January 2008 to January 2010. All cell extracts and 3 g of biotinylated MALAT1 5 -UTR RNA probe. fi m patients underwent standard resection without receiving chemo- The reaction mixture's nal volume was adjusted to 100 L with therapy or radiotherapy preoperatively or postoperatively. The RNA mobility shift buffer (5 mmol/L HEPES, 40 mmol/L KCl, patients' detailed clinicopathologic characteristics are listed in 0.1 mmol/L EDTA, 2 mmol/L MgCl2, 2 mmol/L dithiothreitol, Supplementary Table S1. 1 U RNasin and 0.25 mg/mL heparin). The mixture was incubated for 15 minutes at 30C, and then added to 400 mL of Streptavidin MagneSphere Paramagnetic Particles (Promega) for binding at RNA isolation, reverse transcription, and quantitative real-time room temperature for 10 minutes. Then, the RNA–protein com- PCRqRT-PCR plexes were washed seven times with the RNA mobility shift buffer RNA isolation and the PCR amplification conditions were without heparin. After washing, 30 mLof2ÂSDS-PAGE sample followed as previously described (18). Quantitative real-time buffer was added to the reaction mixture. The retrieved proteins (qRT-PCR; SYBR Green Assay, Roche Diagnostics GmbH) was were detected using standard Western blot analysis. performed on Applied Biosystems 7500 Real-Time PCR System. The relative expression levels of mRNAs were calculated and ÀDD quantified using the 2 CT method after normalization for the RNA-binding immunoprecipitation expression of the control, and GAPDH served as the endogenous The cells (1 107) were lysed with 500 mL of lysis buffer (10 control. The primer sequences were designed by Primer 5.0 and mmol/L Tris-HCl pH 7.4, 1 mmol/L MgCl2, 1 mmol/L EGTA, purchased from Invitrogen. 0.5% CHAPS, 10% glycerol, 0.1 mmol/L PMSF, 5 mmol/L 2-ME) for 20 minutes on ice.