1017 Original Article Page 1 of 11 TLE2 is associated with favorable prognosis and regulates cell growth and gemcitabine sensitivity in pancreatic cancer Shixiong Hu1,2, Zhengbo Chen2, Jinling Gu2, Liyang Tan2, Meifeng Zhang2, Weidong Lin3 1The First Affiliated Hospital of Jinan University, Guangzhou, China; 2Department of General Surgery, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, China; 3The Second People’s Hospital of Foshan, Affiliated Foshan Hospital of Southern Medical University, Foshan, China Contributions: (I) Conception and design: W Lin; (II) Administrative support: WD Lin ; (III) Provision of study materials or patients: SX Hu; (IV) Collection and assembly of data: Z Chen, L Tan, M Zhang; (V) Data analysis and interpretation: Z Chen, J Gu, M Zhang; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Prof. Weidong Lin. The Second People’s Hospital of Foshan City, Guangdong Province, 78 Weiguo Road, Chancheng District, Foshan, China. Email: [email protected]. Background: The transducin-like enhancer of split (TLE) proteins are a group of transcriptional corepressors. They play a crucial role in cellular homeostasis and are involved in various cancers. Compared with other TLE family members, little is known about the role and the underlying mechanism of TLE2 in human cancers. This study aimed to investigate the role of TLE2 in pancreatic ductal adenocarcinoma (PDAC) using in silico analysis and in vitro experiments. Methods: Data were obtained from the Cancer Genome Atlas (TCGA) database to evaluate the prognostic value of TLE2 in PDAC. The MiaPaCa-2 cell line was transfected with siRNA to inhibit endogenous TLE2 expression, and a PANC-1 cell line with stable TLE2 overexpression was constructed using lentiviral transfection, which were confirmed by real-time polymerase chain reaction and western blotting. MTT assay, transwell invasion assays, and flow cytometry were carried out to assess cell viability, invasion, and apoptosis, respectively. TLE2 expression in PDAC cells was altered to evaluate their sensitivity to gemcitabine. Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to predict the biological role of TLE2. Results: High expression of TLEs was significantly associated with increased overall survival (OS) and disease-free survival (DFS) in patients with PDAC. Among the PDAC cell lines, TLE2 expression was lowest and highest in PANC-1 cells and MiaPaCa-2 cells, respectively. TLE2 overexpression impaired the proliferation ability of PANC-1 cells and downregulation of TLE2 promoted the proliferation of MiaPaCa-2 cells. Upregulation of TLE2 in PANC-1 cells induced S-phase accumulation and sensitivity to gemcitabine. In contrast, the downregulation of TLE2 in MiaPaCa-2 cells promoted resistance to gemcitabine. Moreover, bioinformatics analysis also revealed the potential tumor suppressor role of TLE2 and uncovered a close relationship between TLE2 expression and cell cycle regulation. Conclusions: Our results suggest that TLE2 expression is correlated with prognosis in patients with PDAC and show that TLE2 plays a central role in the regulation of cell proliferation, the cell cycle, and gemcitabine sensitivity. This study provides new insights and evidence that TLE2 functions as a tumor suppressor gene and prognostic marker in PDAC. Keywords: Pancreatic cancer; transducin-like enhancer of split family member (TLE family member); transcriptional corepressor; gemcitabine; biomarker Submitted Jun 06, 2020. Accepted for publication Aug 12, 2020. doi: 10.21037/atm-20-5492 View this article at: http://dx.doi.org/10.21037/atm-20-5492 © Annals of Translational Medicine. All rights reserved. Ann Transl Med 2020;8(16):1017 | http://dx.doi.org/10.21037/atm-20-5492 Page 2 of 11 Hu et al. TLE2 as a tumor suppressor in pancreatic cancer Introduction (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) with 10% fetal bovine serum Pancreatic ductal adenocarcinoma (PDAC) is the most (HyClone) at 37 with 5% CO . common pancreatic malignancy and one of the most 2 challenging cancers to treat (1). Despite the substantial ℃ therapeutic advancements, the prognosis for patients Transfection of siRNA with PDAC is still dismal (2-4). Therefore, a better MiaPaCa-2 cells were transfected with siRNAs targeting understanding of the molecular biology and tumor genetics the sequence 5'-CTCCAGAAATAACAAATACATCG-3' of PDAC, and new therapeutic targets that can improve of TLE2. A scramble (5'-GGACGGAGCAGTACAA-3') patient outcomes are urgently needed. without known targets was chosen as a non-sense Transcriptional corepressors are a group of proteins that control. Transfection was performed with Lipofectamine play vital roles in the transcriptional regulation of various 2000 (Thermo Fisher Scientific, USA) in line with genes (5,6). The Groucho (Gro)/transducin-like enhancer the manufacturer’s protocol. siRNA and control of split (TLE) proteins are a large family of gene regulators oligonucleotides were synthesized by Jennio Biological that are widely expressed in eukaryotes and influence the Technology (Guangzhou, China). transcriptional activity of numerous genes (7). Instead of directly binding to DNA strands, TLE proteins bind to other transcription factors (8,9), usually resulting in Lentiviral transfection for TLE2 overexpression transcriptional repression (10,11). TLE proteins have been The TLE2 overexpression lentiviral vector was established observed to interact with transcriptional factors, including by recombining the pGC-LV-GV287-GFP vector with Hes, c-Myc, and Runx (12,13). Additionally, some studies the TLE2 gene. PANC-1 cells overexpressing TLE2 were have reported that TLE1 and TLE3 can participate in constructed via lentiviral-mediated plasmid transfection tumor development as tumor suppressor genes (14-17). (MOI =10). An empty vector served as a transfection TLE2 is a member of the TLE gene family located control. Untransfected cells were used as a normal control. on chromosome 19p13.3. Previous studies have revealed TLE2 expression was validated by quantitative reverse that TLE2 participates in the development of pancreatic transcription polymerase chain reaction (qRT-PCT) and tissue through its interactions with a series of transcription western blot analysis. factors (18). Furthermore, TLE2 is involved in the ventral telencephalon from the early neural plate stage during neurogenesis (19). Many genes known to be involved in RNA extraction and qRT-PCR the regulation of histogenesis and tissue development, Total RNA was extracted from cells using TRIZOL such as Robo and Wnt, may also perform pivotal roles reagent (Invitrogen), and cDNA was reverse transcribed in neoplastic conditions (20-22). Currently, the role with the cDNA Reverse Transcription Kit (Takara TLE2 plays in most human cancers, especially pancreatic Biotechnology). Then, qRT-PCR was performed on cancer, is a long way from being elucidated. Therefore, each cDNA template in 20-μL reaction systems using this study aimed to examine the prognostic value and the SYBR Green Kit (Takara, Japan) on an ABI 7500HT cellular function of TLE2 in PDAC through in silico and Fast real-time PCR system. The following primers in vitro study for the first time, in order to explore the were used for the amplification of TLE2 fragments: role of TLE2 in PDAC and to elucidate the underlying forward 5'-TATTTCCGATTACGGCACTCG-3'; mechanisms. We present the following article in reverse 5'-CTACTCTCCATTCCGACCGC-3', accordance with the MDAR reporting checklist (available GAPDH was used as internal control (forward at http://dx.doi.org/10.21037/atm-20-5492). 5'-TTGTCTCCTGCGACTTCAACAG-3'; reverse 5'-GGTCTGGGATGGAAATTGTGAG-3'). Methods Cells and reagents Western blot analysis PANC-1 and MiaPaCa-2 human pancreatic cancer cell lines Pancreatic cancer cell lines were washed and resuspended were obtained from American Type Culture Collection with PBS twice. Total protein was isolated and quantified © Annals of Translational Medicine. All rights reserved. Ann Transl Med 2020;8(16):1017 | http://dx.doi.org/10.21037/atm-20-5492 Annals of Translational Medicine, Vol 8, No 16 August 2020 Page 3 of 11 using RIPA extraction buffer and the BCA kit (Pierce). The cancers were analyzed according to the transcriptome data protein samples were separated by 10% SDS-PAGE and and clinical parameters obtained from The Cancer Genome transferred onto polyvinylidene fluoride (PVDF) membranes. Atlas (TCGA; http://cancergenome.nih.gov/), which The membranes were then blocked with buffer containing were analyzed with transcripts per million (TPM) as log2 5% non-fat milk and incubated with TLE2 antibody (rabbit (TPM +1) in Gene Expression Profiling Interactive Analysis polyclonal anti-human, Abcam, USA; 1:200, No. ab206147). (GEPIA; http://gepia.cancer-pku.cn) portal (23). Anti-rabbit IgG was used as the secondary antibody (Santa Cruz Biotechnology, No. sc-2357). The blots were visualized Functional annotation analysis with an ELC kit (Millipore, USA). The RNA-seq data (FPKM, fraction per kilobase per million reads) of PDAC were downloaded from the TCGA Cell viability assays portal (TCGA-PAAD). The correlation between the For the evaluation of cell proliferation, cell viability assays expressions of TLE2 and other genes at the transcriptional were carried out. Briefly, different groups of PANC-1 cells level (FPKM) was estimated by