关键词: 食管肿瘤, DEAD-box RNA解旋酶39A, 生物信息学, 信号通路, 细胞侵袭

Abstract:

Objective To discuss the effect of DEAD-box RNA helicase 39A（DDX39A） gene silencing on the proliferation，migration and invasion of the esophageal cancer TE-1 cells，and to clarify its possible mechanism. Methods For bioinformatics analysis， GSE63941， GSE77861， GSE20347， and GSE16153 chip data were downloaded from the GEO database. The esophagel cancer-related data were selected from the TCGA Database.R software was used to analyze the differentially expressed genes.STRING Database was used to construct the protein-protein interaction （PPI） network.Identification of key genes of high relevance was achieved using the MCODE plugin in Cytoscape.The expression of key genes in normal esophageal tissue and esophageal cancer tissue were analyzed with the GEPIA 2 database. Kaplan-Meier Plotter was used to perform survived analysis and plotting for the screened key genes.Cytological experiments were carried out on esophageal cancer TE-1 cells， and small interfering RNA （siRNA）technology was used to silence the expression of DDX39A gene.The TE-1 cells in the logarithmic growth phase were selected and divided into blank （MOCK） group， negative control （si-NC） group， and silencing （si-DDX39A） group. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of DDX39A mRNA and protein in the TE-1 cells in various groups；CCK-8 assay was conducted to detect the proliferation activity of cells in various groups， and the cell scratch assay was used to measure the migration rate of cells in various groups； Transwell chamber assay was used to detect the number of invasion cells in various groups；Western blotting method was used to detect the expression levels of β-catenin， glycogen synthase kinase-3β（GSK3β）， phosphorylated glycogen synthase kinase-3β（p-GSK3β）， c-MYC， Cyclin D1 and nuclear β-catenin proteins in the cells in various groups. Results Analyses using TCGA database combined with the GEO Database yielded a total of 56 differentially expressed genes. MCODE plugin in Cytoscape software identified 41 key genes of high relevance； DDX39A was screened by analyzing 41 genes through the GEPIA 2 and Kaplan-Meier plotter Databases. The results of RT-qPCR and Western blotting methods showed that compared with si-NC group， the expression levels of DDX39A mRNA and protein in the cells in si-DDX39A group were decreased （P<0.05）. The CCK-8 results showed that the proliferation activity of the cells in si-DDX39A group was lower than that in si-NC group （P<0.05）. The cell scratch assay results showed that the cell migration rate in si-DDX39A group after 24 h was lower than that in si-NC group （P<0.05）.The results of Transwell chamber assay showed that the number of invasion cells in si-DDX39A group was lower than that in si-NC group （P<0.05）. Compared with si-NC group， the expression levels of β-catenin， p-GSK3β， c-MYC， Cyclin D1， and nuclear β-catenin in the TE-1 cells in si-DDX39A-1 group and si-DDX39A-3 group were decreased （P<0.01）， but the expression levels of GSK3β protein had no significant differences （P>0.05）. Conclusion Silencing of DDX39A gene could inhibit the proliferation， migration and invasion of TE-1 cells， and the mechanism may be related to the regulation of Wnt/β-catenin signaling pathway.

Key words: Esophageal neoplasms, DEAD-box RNA Helicase 39A, Bioinformatics, Signaling pathway, Cell invasion