| Backgrounds Esophageal squamous cell carcinoma(ESCC) is the most common type of esophageal carcinoma and remains the leading cause of cancer-related death worldwide. DEAD-box RNA helicases play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and neoplastic transformation. DDX46 belongs to DEAD-box helicase family, the expression pattern of DDX46 in ESCC tissues and the biologic role in ESCC progression have not been implicated previously.Aims The aims of present study were to explore the DDX46 expression in human ESCC tissues and cells lines, discover the relationship between DDX46 and ESCC, and elucidate their molecular mechanism, in order to develop novel diagnostic and prognostic methods and potential therapeutic targets.Methods DDX46 expression in human ESCC and adjacent normal tissues were explored using immunohistochemistry, and ESCC cells lines compared with normal esophageal epithelium cell were quantified using q RT-PCR. Next, Lentivirus-mediated RNA interference was applied to silence DDX46 in TE-1 and Eca-109. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Using xenograft model in nude mice to test whether knockdown of DDX46 influenced the tumorigenicity of ESCC cells in vivo. Xenograft tumors were examined twice weekly, length and width measurements were obtained with calipers and tumor volumes were calculated. In vivo bioluminescence imaging was explored. On day 31 after inoculation, animals were euthanized and tumors were excised and weighed. Differentially expressed genes between DDX46-sh RNA-LV group and Control-LV group in TE-1 cells were detected by Affymetrix Gene Chip® Prime ViewTM Human Gene Expression Array, and verified by q RT-PCR and Western blot analysis. The results were analyzed by bioinformatics using Ingenuity® pathway analysi(IPA). Further, the Stress and Apoptosis Signaling Antibody Array Kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown.Results The results showed that DDX46 was significantly upregulated in human ESCC tissues and cell lines(TE-1, Eca-109, TE-11 and KYSE-150), compared with the matched adjacent non-tumor tissues and normal esophageal epithelium cell line Het-1A. DDX46 knockdown led to decreased proliferation, increased the percentage of G1-phase cells and decreased the percentage of S-phase cells, and increased the percentage of apoptotic cells in TE-1 and Eca-109 cells. DDX46 sh RNA-treated xenograft tumors were much smaller than control sh RNA-treated tumors. Tumor volume growth curves revealed that the growth of xenograft tumor in nude mice was significantly slower in DDX46 sh RNA-treated nude tumors than control sh RNA-treated tumors throughout the study period. Knockdown of DDX46 reduced the tumorigenicity of ESCC cells. The gene chip result showed that 1006 genes were differentially expressed by at least 1.5 folds and P < 0.01 in DDX46-sh RNA-LV group compared with Control-LV, including 362 upregulated and 644 downregulated ones. Bioinformatics analysis and gene co-expression network building by IPA identified 6 genes(CD44, DDIT3, ERBB3, FGF2, IL6 ST and OGG1) with specific expression in DDX46-sh RNA-LV with Control-LV group, and this result was confirmed by q RT-PCR and Western blot analysis. There were 5 inhibited signaling pathways and 1 activated signaling pathway, and PI3 K was the molecule non-normally expressed. Western blot analysis results showed that PI3 K expression levels were significantly reduced after DDX46 silencing in TE-1 cells. Moreover, DDX46 silencing leaded to decreased phosphorylation of Akt and IκBα, and increased expression of cleaved Caspase-3, as well as negative regulation of NF-κB signaling pathway.Conclusion These results demonstrate that DDX46 was overexpressed in human ESCC tissues and cell lines, targeted silencing of DDX46 gene inhibited cell proliferation, arrested cell cycle in G0/G1 phase, and induced apoptosis; targeted silencing of DDX46 gene reduced the tumorigenicity of ESCC cells in nude mice. Furthermore, DDX46 silencing probably by inhibiting the activity of PI3 K, incorporated into and downregulated the PI3K/Akt signaling pathway, then downregulated the downstream NF-κB signaling pathway and m TOR signaling pathway, and play the roles of inhibiting ESCC cell proliferation and inducing apoptosis.Integrin signaling pathway was involved via PI3 K and “Cross Talk†with PI3K/Akt signaling pathway. Whether Rho GDI signaling pathway was involved by downregulated PI3 K protein expression following DDX46 silencing, and then suppressed the invasion and metastasis of ESCC, is the goal of the future research. |
