The RNA-Binding Protein DDX18 Promotes Gastric Cancer by Affecting the Maturation of MicroRNA-21

Abstract

Objectives: The noncoding RNAs (ncRNAs) play important roles in gastric cancer. Most studies have focused on the functions and influence of ncRNAs, but seldom on their maturation. DEAD box genes are a family of RNA-binding proteins that may influence the development of ncRNAs, which attracted our attention. By combining a small sample for high-throughput gene microarray screening with large samples of The Cancer Genome Atlas (TCGA) data and our cohort, we aimed to find some gastric cancer-related genes. We evaluated the clinical significance and prognostic value of candidate gene DDX18, which is overexpressed in gastric cancer tissues. To provide a theoretical basis for the development of new therapeutic targets for the treatment of gastric cancer, we investigated its effect on the malignant biological behavior of gastric cancer in vitro and in vivo, and also discuss its mechanism of action. Methods: (i) The differential profiling of mRNA expression in five pairs of gastric cancer and adjacent normal tissues was studied by Arraystar Human mRNA Microarray. By combining this with TCGA data and our cohort, we finally filtered out DDX18, which was upregulated in gastric cancer tissues, for further investigation. (ii) The protein expression of DDX18 was detected by immunohistochemistry staining. Then the relationship between the DDX18 expression level and the clinicopathological data and prognosis was analyzed. (iii) A CCK-8 assay and colony formation assay were used to evaluate the effect of DDX18 on cell growth and proliferation in vitro. A transwell assay was also performed to examine the migration and invasion of gastric cancer cells. Cell apoptosis was analyzed by using a fluorescein isothiocyanate–annexin V/propidium iodide double-staining assay. To identify the role of DDX18 in the tumorigenic ability of gastric cancer cells in vivo, we also established a subcutaneous gastric cancer xenograft model. Coimmunoprecipitation, small RNAseq, and western blotting were performed to explore the mechanism of action of DDX18 in gastric cancer. A patient-derived xenograft (PDX) model was used to confirm the effect of DDX18 in gastric cancer tissues. Result: (i) DDX18 was upregulated in gastric cancer tumor tissues from a TCGA database and our cohort. The expression of DDX18 was also closely related to tumor volume, Borrmann classification, degree of tumor differentiation, cancer embolus, lymph node metastasis, and TNM stage. (ii) DDX18 could promote cell proliferation, migration, and invasion and inhibit cell apoptosis in vivo and in vitro. (iii) DDX18 could promote the maturation of microRNA-21 through direct interaction with Drosha, decreasing PTEN, which could upregulate the AKT signaling pathway. (iv) The PDX model showed that DDX18 could promote the proliferation of gastric cancer tissues by means of the PTEN–AKT signaling pathway. Conclusions: (i) DDX18 can be treated as a molecular marker to assess the prognosis of patients with gastric cancer. (ii) DDX18 could be a potential therapeutic target in gastric cancer.