Effect Of Silencing DDX46 On Migration And Invasion In Tumor Cells Of Human Esophageal Squamous Cell Carcinoma

BACKGROUNDS: Esophageal carcinoma is one of the most common malignant tumors in the digestive system.The overall treatment effect is not ideal to improve the prognosis of patients,which is closely related to the invasion and metastasis of tumor cells and other malignant behavioral processes.RNA helicase plays an important role in the metabolic activities of RNA.Recent studies have shown that RNA helicase is abnormally expressed in a variety of tumors,which is involved in the regulation of malignant behavior of tumors and is related to the prognosis of patients.As a member of the RNA helicase family,DDX46 gene is highly expressed in the tissues and cells of esophageal squamous cell carcinoma.Knockdown DDX46 gene can inhibit the proliferation of esophageal squamous cell carcinoma cells and induce apoptosis,but the correlation between DDX46 gene and malignant behavior of esophageal squamous cell carcinoma cells invasion and metastasis has not been reported.OBJECTIVES: Knock down the expression of DDX46 gene in esophageal squamous cell carcinoma by RNAi technology to observe the effect of DDX46 gene on the invasion and migration ability of esophageal squamous cell carcinoma,explore the role and possible molecular mechanisms of DDX46 genes in the invasion and metastasis of esophageal squamous cell carcinoma,with a view to providing an experimental basis for finding new potential molecular markers and therapeutic targets for esophageal squamous cell carcinoma.METHODS: The human esophageal squamous cell carcinoma cell lines(Eca-109 and TE-1)were transfected with green fluorescent labeled lentivirus using RNAi technique.The expression of DDX46 gene was reduced in experimental group(shDDX46 group)and Control group(shCtrl group)was the target cells of empty vector transfection.Sevety-two hours after transfection,the cell transfection efficiency wasjudged according to the expression rate of green fluorescent protein in the target cell.Quantitative real-time polymerase chain reaction(qRT-PCR)detected the knockdown efficiency of the target cell DDX46 gene at mRNA level,and Western Blot detected the knockdown efficiency of DDX46 protein expression level.Wound healing,invasion assay and migration assay were performed to detect the changes of invasion and migration ability.Western Blot was used to detect changes of fibronectin,and Affymetrix GeneChip detected differentially expressed genes in TE-1 cells.The results of differentially expressed genes were analyzed by Ingenuity pathway analysis online analysis tool.Finally,combining literature to explore the role and possible molecular mechanism of DDX46 on the invasion and migration process for esophageal squamous cell carcinoma.RESULTS: shRNA lentivirus infected target cells for 72 hours,the cells were observed to be in good condition under the microscope,and the cell infection efficiency reached more than 80%.Knockdown expression of DDX46 at both mRNA and protein level were detected after shRNA transfection,and knockdown efficiency of DDX46 gene mRNA level reached 84.7%(Eca-109)and 88.2%(TE-1).Western Blot results showed that DDX46 protein expression was significantly inhibited in the target cells,subsequent experiments were allowed.Wound healing results showed that the average cell mobility of Eca-109 in shDDX46 group(0.21±0.01)was significantly lower than that in shCtrl group(0.31±0.02)(P<0.01),and shDDX46 group(0.41±0.04)was significantly lower than shCtrl group(0.61±0.02)(P<0.01)in TE-1 cells.Invasion assay results showed that the number of invasive matrix Eca-109 cells in the shDDX46 group(40±1.68)was lower than that in the shDDX46 group(54±5.24),the average invasion and metastasis rate in the shDDX46 group(0.74±0.03)was lower than that in the shCtrl group(1.00±0.10);The number of invasive matrix TE-1 cells in the shDDX46 group(42±3.56)was lower than that in the shDDX46 group(93±6.45),the mean invasion and metastasis rate of shDDX46 group(0.46±0.04)was lower than that of shCtrl group(1.00±0.07),and the differences were statistically significant(P<0.05).In transwell assay,the number of migratory cells of Eca-109 in shDDX46 group(68±3.12)decreased compared with shCtrl group(87±4.03)after 30h(P<0.01),the cell migration rate of shDDX46 group(0.78±0.04)decreased compared with shCtrl group(1.00±0.05)(P<0.01);The number of migratory cells of TE-1 in shDDX46 group(81±8.08)decreased compared with shCtrl group(170±1.43)after 24h(P<0.01),the cell migration rate of shDDX46 group(0.48±0.05)was lower than shCtrl group(1.00±0.01)(P<0.01).Expression of fibronectin in TE-1 cells decreased after DDX46 gene was knocked down.IPA differentially expressed genes showed that the integrin signaling pathway was significantly inhibited.CONCLUSION: DDX46 plays an essential role in invasion and migration of human esophageal squamous cell carcinoma,shRNA interfered with DDX46 gene can significantly inhibit the invasion and metastasis ability.Knockdown DDX46 genes may inhibit the activity of focal adhesion kinase by downregulating integrin pathway signaling.DDX46 may be a potential molecular marker and therapeutic target for esophageal squamous cell carcinoma.