DEAD-box helicase 56 functions as an oncogene promote cell proliferation and invasion in gastric cancer via the FOXO1/p21 Cip1/c-Myc signaling pathway

DEAD-box helicase (DDX) family exerts a critical effect on cancer initiation and progression through alternative splicing, transcription and ribosome biogenesis. Increasing evidence has demonstrated that DEAD-box helicase 56 (DDX56) is over-expressed in several cancers, which plays an oncogenic role. Till the present, the impact of DDX56 on gastric cancer (GC) remains unclear. We conducted high-throughput sequencing (RNA-seq) to demonstrate aberrant DDX56 levels within 10 GC and matched non-carcinoma tissue samples. DDX56 levels were detected through qRT‐PCR, western blotting (WB) and immunochemical staining in GC patients. We conducted gain- and loss-of-function studies to examine DDX56’s biological role in GC development. In vitro, we carried out 5‑Ethynyl‑2‑deoxyuridine (EdU), scratch, Transwell, and flow cytometry (FCM) assays for detecting GC cell growth, invasion, migration and apoptosis. Additionally, gene set enrichment analysis (GSEA), WB assay, and Encyclopedia of RNA Interactomes (ENCORI) were carried out for analyzing DDX56-regulated downstream genes and signaling pathways. In vivo, tumor xenograft experiment was performed for investigating how DDX56 affected GC development within BALB/c nude mice. Functionally, DDX56 knockdown arrested cell cycle at G1 phase, invasion and migration of AGS and MKN28 cells, and enhanced their apoptosis. Ectopic DDX56 expression enhanced the cell growth, migration and invasion, and inhibited apoptosis. Knockdown of DDX56 suppressed GC growth in the tumor models of BALB/c nude mice. Mechanistically, DDX56 post-transcriptionally suppressed FOXO1/p21 Cip1 protein expression, which could activate its downstream cyclin E1/CDK2/c-Myc signaling pathways. This sheds lights on the GC pathogenic mechanism and offers a potential anti-cancer therapeutic target.

DEAD-box解旋酶（DEAD-box helicase, DDX）家族可通过可变剪接、转录调控及核糖体生物发生，对癌症的发生与进展发挥关键调控作用。越来越多研究证据表明，DEAD-box解旋酶56（DEAD-box helicase 56, DDX56）在多种癌症中呈异常高表达，并发挥致癌功能。截至目前，DDX56对胃癌（gastric cancer, GC）的具体影响仍未明确。本研究通过高通量转录组测序（RNA-seq），分析了10对胃癌组织及配对癌旁正常组织中DDX56的表达水平异常情况。随后采用实时荧光定量聚合酶链反应（quantitative real-time PCR, qRT-PCR）、蛋白质印迹（western blotting, WB）及免疫组织化学染色，检测了胃癌患者组织中的DDX56表达水平。为探究DDX56在胃癌发生发展中的生物学功能，本研究开展了功能获得与功能缺失实验。体外实验中，我们通过5-乙炔基-2'-脱氧尿苷（5-Ethynyl-2-deoxyuridine, EdU）实验、划痕实验、Transwell实验及流式细胞术（flow cytometry, FCM），分别检测胃癌细胞的增殖、侵袭、迁移与凋亡情况。此外，本研究借助基因集富集分析（gene set enrichment analysis, GSEA）、蛋白质印迹实验及RNA互作组百科全书（Encyclopedia of RNA Interactomes, ENCORI）数据库，分析了DDX56调控的下游基因及相关信号通路。体内实验中，我们构建BALB/c裸鼠异种移植瘤模型，探究DDX56对胃癌体内生长的影响。功能实验结果显示：敲低DDX56可使AGS和MKN28细胞的细胞周期阻滞于G1期，抑制其侵袭与迁移能力，并促进细胞凋亡；而过表达DDX56则可增强细胞增殖、迁移及侵袭能力，同时抑制细胞凋亡。在裸鼠移植瘤模型中，敲低DDX56可显著抑制胃癌移植瘤的生长。机制层面，DDX56可通过转录后调控抑制FOXO1/p21 Cip1蛋白的表达，进而激活下游细胞周期蛋白E1/细胞周期蛋白依赖性激酶2/c-Myc信号通路。本研究揭示了胃癌发生的潜在致病机制，为胃癌的抗癌治疗提供了全新的潜在靶点。