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@#Objective To investigate the relationship between DDX46 genes and invasion and migration of esophageal squamous cell carcinoma cells. Methods Human esophageal squamous cell carcinoma cells TE-1 were transfected by fluorescent marker shRNA lentivirus (shDDX46 group), and an empty vector was transfected as a control (shCtrl group). The expression rate of green fluorescent protein under the microscope was used to evaluate the cell transfection efficiency. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blotting (WB) detected the knockdown efficiency of the target gene at the mRNA and protein expression levels. Wound healing, invasion assay and migration assay detected the changes of invasion and metastasis ability. Classical pathway analysis was used to explore signaling pathway changes and the possible mechanism of DDX46 in the invasion and metastasis was explored by detecting fibronectin expression. Results DDX46 gene at mRNA and protein levels was significantly inhibited after lentiviral transfection. Wound healing showed that after 8 h the cell mobility of TE-1 cells decreased significantly (P=0.001). Invasion assay showed that after 24 h the average cell metastasis rate of TE-1 cells was lower in the shDDX46 group than that in the shCtrl group (P<0.001). The cell metastasis rate in the shDDX46 group corresponding to observation points in the transwell assay was lower than that in the shCtrl group (P<0.001) after 24 h culture. The results of the classical pathway analysis showed that the integrin signaling pathway activity was inhibited, further exploration of the mechanism of action found that the expression of fibronectin associated with cell adhesion was decreased. Conclusion DDX46 gene is related to the invasion and migration ability of esophageal squamous cell carcinoma cells. Knockdown of DDX46 genes may reduce cell adhesion by downregulating the integrin pathway signaling.
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@#Objective To explore the mechanism of DDX46 regulation of esophageal squamous cell carcinoma. Methods Picture signals of fluorescence in gene array were scanned and differential expression of gene in two groups (a DDX46-shRNA-LV group and a control-LV group) were compared by GCOSvL.4 software. These differential expressed genes were analyzed by bioinformatics methods finally, and validated by quantitative real time polymerase chain reaction (qRT-PCR) analysis. Results According to the screening criteria of fold change ≥2 and P<0.05, 1 006 genes were differentially expressed after DDX46 knockdown, including 362 up-regulated and 644 down-regulated genes. Bioinformatics analysis and gene co-expression network building identified that these differentially expressed genes were mainly involved in cell cycle, proliferation, apoptosis, adhesion, energy metabolism, immune response, etc. Phosphatidylinositol 3-kinase (PI3K) was the key molecule in the network. The results of RT-qPCR were completely consistent with the results of gene microarra. Conclusion Bioinformatics can effectively exploit the microarray data of esophageal squamous cell carcinoma after DDX46 knockdown, which provides a valuable clue for further exploration of DDX46 tumorigenesis mechanism and helps to find potential drug therapy.
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Objective To investigate the expression of DDX46 protein in colorectal cancer and its correlation with expressions of C-erbB-2, Ki-67, p53 and nm23 proteins. Methods A total of 149 cases of colorectal cancer tissues and 45 adjacent mucosa tissues after operation in the Wuxi No.2 Hospital Affiliated to Nanjing Medical University from January 2010 to December 2012 were collected. The expressions of DDX46 protein and C-erbB-2, Ki-67, p53 and nm23 proteins in tissues of colorectal cancer were detected by using immunohistochemistry. The relations between the expression level of DDX46 protein and the clinicopathological features including the age, gender, tumor differentiation, tumor infiltration, lymph node metastasis, TNM stage were analyzed, and the relations between DDX46 protein expression and expressions of C-erbB-2, Ki-67, p53 and nm23 proteins were also analyzed. Results Of 149 colorectal cancer tissues, 50 (33.6％) showed low expressions of DDX46 protein and 99 (66.4％) showed high expressions. Of 45 adjacent mucosa tissues, 37 (82.2％) showed low expressions of DDX46 protein, and 8 (17.8％) showed high expressions. There were significant differences between the high expression rate of DDX46 protein in cancer tissues and adjacent mucosa tissues (χ2=33.09, P<0.01). There were statistical differences in the expression level of DDX46 protein and tumor differentiation, tumor infiltration, lymph node metastasis, TNM staging (all P<0.05). The ratio of high-expressed DDX46 protein was increased with the low differentiation, high invasion degree, lymph node metastasis and late TNM staging. DDX46 protein expression was correlated with expressions of Ki-67, p53 and nm23 proteins (all P< 0.05), and the expression of DDX46 protein had a positive correlation with expressions of Ki-67 and p53 proteins (r values were 0.161 and 0.347), and a negative correlation with expression of nm23 protein (r= -0.561). Conclusions The high expression of DDX46 protein in colorectal cancer tissues is related with malignant biological behavior of the tumors and the key oncogenes. DDX46 protein could be regarded as the potential marker for the diagnosis and molecular targeting therapy of colorectal cancer.
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@#Objective To observe the growth of xenografted tumor in nude mice after DDX46 expression decreased, and to further study the role of DDX46 in the development and progression of esophageal squamous cell carcinoma. Methods DDX46-shRNA mediated RNAi was applied to silencing DDX46 in Eca-109 cells. Twenty-five female BALB/c nude mice were divided into 3 groups: an experiment group (DDX46-shRNA-LV, n=10), a control group (Control-LV, n=10) and a blank control group (Het-1A, n=5). The prepared Eca-109 cells of DDX46-shRNA-LV and Control-LV were subcutaneously injected into the right armpit of mice (4×106 cells per mouse), while Het-1A cells were subcutaneously injected into the bilateral armpits of mice (4×106 cells per side). Tumor growth was monitored twice a week on the 14th day after injection. Tumor volume was measured with calipers, in vivo imager to observe the fluorescence of each group. Further, western blotting analysis was used to detect the changes of apoptosis signaling molecules in xenografted tumor after DDX46 silence. Results The growth of xenografted tumor in nude mice was significantly slower in the DDX46-shRNA-LV group than that in the Control-LV group throughout the study period (P<0.001). Western blotting analysis showed that silencing DDX46 effectively suppressed the expression of DDX46, and upregulated the expression of cleaved Caspase-3 and cleaved PARP-1 in xenografted tumor (P<0.01). Conclusion DDX46 is involved in the development and progression of esophageal squamous cell carcinoma, and the silence of DDX46 expression can inhibit the growth of esophageal squamous cell carcinoma, which probably by positive regulation of apoptosis signaling pathway.
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Objective To investigate the effect of DDX46 gene on the radiosensitivity of colorectal cancer cells and underlying mechanism.Methods SW480 cells transfected with DDX46 RNAi or its empty plasmid by lentivirus were set as experimental group and control group,respectively.After 72 h of transfection,the cells were irradiated with 4 Gy X rays.The cell viabilities of these two groups were detected by CCK-8 assay.The number of γ-H2AX foci and the expressions of some key proteins related to DNA damage repair were detected by immunofluorescence technique and Western blot at 24 h after irradiation.Results At 24 h after 4 Gy irradiation,the cell vitality of experimental group was decreased to (15.02±3.92)％(t=-4.696,P ＜ 0.05) of control and(17.43 ±1.83)％(t=4.844,P＜0.01) of nonirradiated cells,but there was no significant difference between 4 Gy irradiated control cells and nonirradiated cells.Meanwhile,compared with the control group,the ATM protein expression level (t =7.530,P ＜0.01) and the number of γ-H2AX foci (t =-3.108,P ＜0.05) were significantly increased in the experimental SW480 cells,while the expression levels of p-ATM and Rad50 were significantly reduced (t =4.260,4.260,P ＜ 0.05),and the protein levels of DNA-PK in these two groups had tiny difference.Conclusions DDX46 RNA silence increases the radiation sensitivity of SW480 cells by inhibiting ATM activation and DNA repair.