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Potential Cerna Networks Involved in Autophagy Suppression Of 600 INTERNATIONAL JOURNAL OF ONCOLOGY 54: 600-626, 2019 Potential ceRNA networks involved in autophagy suppression of pancreatic cancer caused by chloroquine diphosphate: A study based on differentially‑expressed circRNAs, lncRNAs, miRNAs and mRNAs DAN-MING WEI1*, MENG-TONG JIANG1*, PENG LIN2, HONG YANG2, YI-WU DANG1, QIAO YU1, DAN-YU LIAO1, DIAN-ZHONG LUO1 and GANG CHEN1 Departments of 1Pathology and 2Medical Ultrasonics, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China Received April 14, 2018; Accepted October 19, 2018 DOI: 10.3892/ijo.2018.4660 Abstract. Autophagy has been reported to be involved in the pivotal roles in the autophagy of pancreatic cancer cells, which occurrence and development of pancreatic cancer. However, lays the theoretical foundation for subsequent investigations the mechanism of autophagy-associated non-coding RNAs on pancreatic cancer. (ncRNAs) in pancreatic cancer remains largely unknown. In the present study, microarrays were used to detect differential Introduction expression of mRNAs, microRNAs (miRNAs), long ncRNAs (lncRNAs) and circular RNAs (circRNAs) post autophagy Pancreatic cancer is a highly aggressive and fatal malignancy, suppression by chloroquine diphosphate in PANC-1 cells. with stably high incidence and mortality for the past 30 years Collectively, 3,966 mRNAs, 3,184 lncRNAs and 9,420 circRNAs globally (1-4). Patients with pancreatic adenocarcinoma account were differentially expressed. Additionally, only two miRNAs for ~85% of all patients with pancreatic cancer and have a poor (hsa-miR-663a-5p and hsa-miR-154-3p) were underexpressed prognosis, compared with patients with other common solid in the PANC-1 cells in the autophagy-suppression group. tumors (2,5-9). Pancreatic cancer is primarily treated through Furthermore, miR-663a-5p with 9 circRNAs, 8 lncRNAs and surgical resection, whereas postoperative patients and patients 46 genes could form a prospective ceRNA network associ- with advanced disease are frequently subjected to adjuvant ated with autophagy in pancreatic cancer cells. In addition, therapies with radiotherapy, chemotherapy, and a combination another ceRNA network containing miR-154-3p, 5 circRNAs, of radiotherapy and chemotherapy that are associated with 2 lncRNAs and 11 genes was also constructed. The potential high recurrence rates and poor treatment effectiveness (10). multiple ceRNA, miRNA and mRNA associations may serve The recent development of and research into molecular targeted drugs have great significance for the early diagnosis and prognosis of malignant tumors, and have ushered a new era of tumor therapy (11-15). However, the overall treatment Correspondence to: Professor Gang Chen or Professor Dian-Zhong effect of these drugs remains poor, and further investigation Luo, Department of Pathology, First Affiliated Hospital of Guangxi is required (16). Medical University, Nanning, Guangxi 530021, P.R. China Autophagy is a process by which eukaryotic cells undergo E-mail: [email protected] self-degradation of intracellular damaged macromolecular E-mail: [email protected] proteins and organelles (17,18). Autophagy at the physiological level is necessary to maintain the stability of * Contributed equally the internal environment, and the occurrence of a variety of diseases, including inflammation, tumors and degenerative Abbreviations: rRNA, ribosomal RNA; tRNA, transfer RNA; diseases, is frequently accompanied by an abnormal snRNA, small nuclear RNA; lncRNA, long non-coding RNA; autophagic level (19-25). Currently, well-characterized, miRNA, microRNA; circRNA, circular RNA; ceRNAs, competitive endogenous RNAs; RIN, RNA integrity number; GO, Gene autophagy-associated proteins include Beclin 1, Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; microtubule-associated-protein-1-light-chain-3 (LC3) and GEO, Gene Expression Omnibus; ATG12, autophagy-related 12 P62, and well-studied pathways include mechanistic target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) Key words: pancreatic cancer, autophagy, ceRNA, circRNA, pathways (26,27). lncRNA, chloroquine diphosphate Recently, the important role of autophagy in tumors has been recognized (19,28). Studies demonstrated that pancreatic cancer cells have an increased autophagy level, compared with WEI et al: ceRNA NETWORKS IN AUTOPHAGY SUPPRESSION OF PANCREATIC CANCER 601 other tumor cells (29-34). The growth of PDAC had a distinct chloroquine diphosphate being frequently used as a classic dependence on autophagy in vivo and in vitro (35). Inhibition autophagic inhibitor (65,66), the following concentration of autophagy is effective for the treatment of pancreatic cancer gradient of chloroquine diphosphate (Sigma-Aldrich; Merck through animal experiments (36). Another study revealed KGaA, Darmstadt, Germany) was designed as recommended by that autophagy can be activated by gemcitabine and ionizing the manufacturer's protocols: 0, 25, 50, 75 and 100 µM. When radiation in the treatment of pancreatic cancer cells, and activated the number of cells in the culture bottle grew to ~5x106, the autophagy serves a role in cancer suppression (37). However, original medium was replaced by a complete DMEM containing whether autophagy promotes the occurrence, development and chloroquine diphosphate. Following treating the cells for 12 h prognosis of pancreatic cancer is controversial (38). Therefore, at 37˚C with the drug, the cellular proteins were immediately the role of autophagy in the occurrence, development, prognosis extracted using Radioimmunoprecipitation Assay Lysis Buffer and treatment of pancreatic cancer and its mechanism requires (Beyotime Institute of Biotechnology, Haimen, China). further examination. mRNA is a single-stranded RNA that is transcribed using Western blotting. The lysis solution (radioimmunoprecipitation one of the DNA chains as the template and carries the genetic assay buffer:phenylmethylsulfonyl fluoride; 100:1, information that guides protein synthesis. Thus, mRNA Beyotime Institute of Biotechnology) was prepared. The occupies an important position in the heredity dogma (39). protein concentration in the lysate was determined with a mRNA serves a key role in various human diseases and is bicinchoninic acid kit (Beyotime Institute of Biotechnology), involved in autophagy pathways (40,41); for example, signal according to the manufacturer's instructions. Western transducer and activator of transcription 3 could inhibit blotting was performed according to routine procedures, autophagy in pancreatic cancer cells (42) and ubiquitin and conducted with a 12% separating gel and a 5% stacking specific peptidase 1 regulates autophagy by targeting Unc‑51 gel. A total of 20 µg protein was added to the glue holes. like autophagy activating kinase 1 (ULK1) (43). Non-coding The electrophoresis condition was set to 120 Ma for 1 h. RNA (ncRNA) refers to RNA that does not encode a protein Following the electrophoresis, the protein was transferred to and includes ribosomal RNA (rRNA), transfer RNA (tRNA), the polyvinylidene (PVDF) membrane (Beyotime Institute small nuclear RNA (snRNA), long ncRNA (lncRNA), of Biotechnology). Subsequently, the PVDF membrane microRNA (miRNA) and circular RNA (circRNA) (44-50). was placed into the Bull Serum Albumin blocking buffer The involvement of lncRNAs and miRNAs in cancer has (Beyotime Institute of Biotechnology) for shaking at 37˚C for been studied intensively, whereas the potential significance of 2 h. Primary antibodies were incubated at 4˚C overnight and circRNAs in tumors has attracted attention over the last two secondary antibodies were incubated at room temperature years (51). The post-transcriptional levels of circRNAs serve a for 2 h. The primary antibodies were LC3 β‑specific rabbit notable regulatory role in gene expression (52). Furthermore, polyclonal (1:1,000; cat. no. 18725-1-AP) and P62 mouse the sponge effect of circRNAs on miRNAs is an important monoclonal antibodies (1:2,000; cat. no. 66184-1-Ig), mechanism (53). circRNAs can contain multiple miRNA which represented the state of autophagy, and the GAPDH binding sites, and attract miRNAs effectively to reduce horseradish peroxidase-labeled internal reference antibody miRNA-mediated mRNA inhibition (54-56). Based on the (1:10,000; cat. no. HRP-60004), andhe secondary antibodies interaction between these three types of ncRNAs and the used were goat anti-mouse IgG (H+L), allopregnanolone current research status in pancreatic cancer, it was considered conjugate (1:1,000; cat. no. SA00002-1) (all from ProteinTech that an investigation of the effect of lncRNAs and circRNAs as Group, Inc., Chicago, IL, USA). The gray scale values of the competitive endogenous RNAs (ceRNAs) on the targeting of protein bands from the raw image were determined using the miRNAs in the development of pancreatic cancer is beneficial. ImageJ 1.48 (National Institutes of Health, Bethesda, MD, Recently, the number of autophagy-associated lncRNAs USA) and the formula Final gray scale value (G) = [G x (target has been determined to be limited (57-60), and investigations band) - G x (background)] / [G x (internal reference band on circRNAs and their associations with autophagy have of the group) - G x (background)]. The data were analyzed not been reported to date. However, due to autophagy being using SPSS software 22 (SPSS, Inc., Chicago, IL, USA), and involved in the occurrence and development of a variety the results were plotted using the GraphPad Prism 5 software of
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