MOLECULAR MEDICINE REPORTS 15: 2633-2642, 2017 Knockdown of ZFPL1 results in increased autophagy and autophagy‑related cell death in NCI‑N87 and BGC‑823 human gastric carcinoma cell lines YONG-ZHENG XIE, WAN-LI MA, JI-MING MENG and XUE-QUN REN Department of General Surgery, Huaihe Hospital of Henan University, Kaifeng, Henan 475000, P.R. China Received October 1, 2015; Accepted September 28, 2016 DOI: 10.3892/mmr.2017.6300 Abstract. Macroautophagy, which will hereafter be referred Introduction to as autophagy, is an evolutionarily conserved process, during which cells recycle and remove damaged organelles Gastric cancer (GC) is the fifth most common cancer and proteins in response to cellular stress. However, the worldwide, and is currently the third leading cause of mechanisms underlying the regulation of autophagy remain cancer-associated mortality. GC is particularly prevalent in to be fully elucidated. The present study demonstrated that Asia (1). Unfortunately, the majority of patients with GC present knockdown of zinc finger protein like 1 (ZFPL1) induces with late stage cancer, and therefore require palliative chemo- autophagy and increases autophagic cell death in NCI-N87 therapy (2). Treatment of GC continues to present a challenge, and BGC-823 human gastric carcinoma cell lines. To particularly with regards to the high mortality-to-incidence examine the role of ZFPL1 in gastric carcinoma cells, ZFPL1 ratio, despite significant progress being made in early detection expression was downregulated by lentiviral infection. Zinc and treatment (3). Genetic alterations are thought to be impor- finger domain-FLAG was used to compete with ZFPL1 tant factors in the vast majority of solid tumors. Recently, gene for golgin A2/GM130 binding. Autophagy was analyzed therapy has been considered an attractive therapeutic option. by red fluorescent protein-microtubule-associated protein In addition, autophagy-related genes have attracted attention 1A/1B-light chain 3 (LC3) puncta, LC3I to LC3II conversion, as novel potential targets in cancer treatment (4,5). and p62 expression. The results demonstrated that knockdown Autophagy is a dynamic process, during which cells of ZFPL1 was able to significantly increase cell death rate. recycle and remove damaged organelles and proteins, in However, ZFPL1 knockdown exerted almost no effect on order to ensure cell survival in response to cellular stress (6). the expression of apoptosis-associated markers, including Furthermore, autophagy can induce cell death under certain B cell lymphoma 2 (Bcl-2), Bcl-x, Bcl-2-associated X protein, conditions. Therefore, the two opposing effects of autophagy BH3 interacting domain death agonist, p53, and the classical serve an important role in cellular differentiation, develop- caspase family members, caspase-3, caspase-8 and caspase-9. mental processes and human disease (7). Several proteins An endogenous ZFPL1‑GM130 association was identified in are involved in the detection of autophagic activity, including NCI-N87 cells and BGC-823 cells by co-immunoprecipitation. microtubule-associated protein 1A/1B-light chain 3 (LC3) and Furthermore, cell death was restricted following treatment the LC3 binding protein, sequestome 1/p62 (8). of ZFPL1 knockdown cells with an autophagy inhibitor. The significance of autophagy in cell cycle progression Therefore, knockdown of ZFPL1 expression may induce cell and cell death has previously been reported. A previous death via autophagy, rather than apoptosis. These results study demonstrated that activated autophagy contributes suggest that ZFPL1 may serve an important role in regulating to matrine-induced cell death of the SGC-7901 GC cell autophagy in NCI-N87 and BGC-823 cells. line (9). Conversely, autophagy-mediated high mobility group box 1 release promotes GC cell survival via receptor for advanced glycation endproducts activation of extracellular signal-regulated kinases 1/2 (10). However, the function of autophagy in GC remains to be elucidated. Previous studies indicated the potential roles of zinc finger proteins in gastric cancer progression and the association with Correspondence to: Dr Xue-Qun Ren, Department of General Surgery, Huaihe Hospital of Henan University, 8 Baobei Road, autophagy (11). The importance of zinc finger protein like 1 Kaifeng, Henan 475000, P.R. China (ZFPL1) in autophagy and human GC resulted in the aim of E-mail: [email protected] the present study; to explore the potential antitumor effects of ZFPL1 knockdown, using short hairpin (sh)RNA against Key words: zinc finger protein like 1, autophagy, cell death, gastric ZFPL1 (shZFPL1). The present study explored whether knock- carcinoma cell down of ZFPL1 would increase autophagy, inhibit protein glycosylation and promote autophagy-related cell death. In addition, the present study evaluated whether the effects of 2634 XIE et al: ZFPL1 KNOCKDOWN INDUCES AUTOPHAGY AND AUTOPHAGY-RELATED CELL DEATH IN GC CELLS ZFPL1 on autophagy correlated with the interaction between current study were obtained using shZFPL1#1; shZFPL1#2 ZFPL1 and GM130. worked similarly. For lentiviral transduction, human embry- onic kidney (HEK)293T cells (Shanghai Ling Feng Chemical Materials and methods Reagent Co., Ltd.) were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with Reagents and antibodies. The MTT Cell Proliferation 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, and Cytotoxicity Assay kit was purchased from Beyotime Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin at Institute of Biotechnology (Haimen, China). Immunoblotting 37˚C in a humidified atmosphere containing 5% CO2. When was performed using anti-ZFPL1 (cat. no. sc-515393; Santa 75-80% confluent, the HEK293T cells were transduced Cruz Biotechnology, Inc., Dallas, TX, USA), anti-β-actin (incubated at 3% CO2, 37˚C, 25 min) with the third‑generation (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.), anti-LC3 packaging plasmids (Cell Signaling Technology, Inc.) pMD2. (cat. no. L8918; Sigma-Aldrich; Merck Millipore, Darmstadt, VSVG, pRSV-REV and pMDLg/pRRE, alongside the transfer Germany), anti-p62 (cat. no. N1163; Sigma-Aldrich; Merck constructs. The fresh supernatant was filtered using a 0.45 µm Millipore), cleaved caspase antibody sampler kit (cat. no. 2855; filter for further use. The first harvest pool was placed into Cell Signaling Technology, Inc., Danvers, MA, USA), anti-RL2 50 ml tubes and stored at 4˚C and then centrifuged for 10 min at (cat. no. SAB1304907; igma-Aldrich; Merck Millipore), anti-p53 1,153 x g to produce a pellet consisting of cells and debris. The (cat. no. ab1431; Abcam, Cambridge, MA, USA), anti-BH3 cell-free supernatant was subjected to ultra-centrifugation for interacting domain death agonist (Bid; cat. no. ab32060; 120 min (16˚C) at 49,460 x g, after which the supernatant was Abcam), anti-B-cell lymphoma 2 (Bcl-2)-associated X protein discarded and the pellet was resuspended in HBS. The vector (Bax; cat. no. 554104; BD Pharmingen, San Diego, CA, USA), stock solutions were pooled to produce a homogenous vector anti-Bcl-x (cat. no. 551269; BD Pharmingen) and anti-Bcl-2 stock solution and stored at -80˚C. Freezing and thawing of (cat. no. 610539; BD Biosciences, San Jose, CA, USA). the lentiviral stock were avoided as much as possible. Infected Horseradish peroxidase (HRP)-conjugated secondary anti- NCI-N87 and BGC-823 cells were rinsed with PBS, and were bodies (cat. no. 58203) were purchased from Cell Signaling allowed to recover for 12 h prior to further experimentation. Technology, Inc. Brefeldin A (BFA) and bafilomycin A1 were purchased from Sigma-Aldrich; TRIzol reagent was obtained Cell growth inhibition assay. Cell growth inhibition was from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, detected using the MTT Cell Proliferation and Cytotoxicity MA, USA); RevertAid First Strand cDNA Synthesis kit was Assay kit (Siemens AG, Munich, Germany). The present purchased from Thermo Fisher Scientific, Inc.; and TransStart study consisted of the following four groups: Control shRNA Green qPCR SuperMix was from Beijing Transgen Biotech group, shZFPL1 group, control shRNA + bafilomycin A1 Co., Ltd. (Beijing, China). All other reagents were of analytical group and shZFPL1 + bafilomycin A1 group. Bafilomycin A1 grade. group cells were incubated with 3 mg/ml bafilomycin A1 at room temperature for 3 h. Briefly, cells in the logarithmic Cell culture and lentiviral infection. The NCI-N87 and growth phase were collected and reseeded into 96-well BGC-823 human GC cell lines were purchased from the plates at 10,000 cells/well. Following a 24 h incubation, the American Type Culture Collection (Manassas, VA, USA). The medium was gently removed from all wells and fresh medium NCI-N87 and BGC-823 cells were cultured in RPMI-1640 containing 10 µl MTT (5 mg/ml) was added to each well. medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented Following a further 4 h incubation at 37˚C, 150 µl dimethyl with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, sulfoxide (Sigma-Aldrich; Merck Millipore) was added, and Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin at 37˚C the plates were slowly agitated for 10 min at room tempera- in a humidified atmosphere containing 5% CO2. Autophagy ture. Subsequently, Sorensen's glycine buffer (0.1 M glycine, was induced by culturing cells in medium supplemented 0.1 M NaCl; pH 10.5) was added, and the absorbance of each with Hanks' balanced salt solution (HBSS; ph 7.2-7.3) or well was measured at 490 nm in 10 min. The cell growth BFA (1 µg/ml) overnight (3% CO2, 37˚C) prior to lentiviral inhibition rate was calculated as follows: (1 - experimental infection. The recombinant plasmids were constructed group absorbance/control group absorbance) x100. A graph according to our previous study (12). Transient transfections was plotted using GraphPad Prism 5 software (GraphPad were performed using FUGENE 6 (Roche Diagnostics, Basel, Software, Inc., La Jolla, CA, USA). All experiments were Switzerland) according to the manufacturer’s instructions. carried out in triplicate. The ZFPL1 overexpression was performed using ZFPL1 mimics (cat.