The aim of the present study was to employ RNA interference

The aim of the present study was to employ RNA interference (RNAi) technology to construct and select shRNA-Nanog recombinant plasmids for the inhibition of Nanog gene expression and transfer these plasmids into the human gastric cancer cell line, SGC-7901, as well as to detect the expression of Nanog and the effects on the proliferation, migration, invasion, cell cycle and apoptosis of SGC-7901 cells. determined by CCK-8 assay. The migration and invasion of the SGC-7901 cells was determined PCI-32765 by Transwell assays, while the cell cycle and apoptosis were analyzed by flow cytometry. The group with the highest inhibition rate was successfully constructed and identified. It was observed that the proliferation, invasion and migration capacity of the cells was reduced, that the cell cycle was arrested at the S phase and that apoptosis was significantly increased. The Nanog gene in gastric cancer cells is closely associated with cell proliferation, the cell cycle, apoptosis and migration and invasion abilities. The present study establishes the foundations for a novel approach for the genetic treatment of gastric cancer. reported that there may be gastric cancer stem cells in the gastric cancer tissues that express Nanog (7). These results demonstrated that the expression level of Nanog in gastric cancer tissues was higher compared with paracancerous tissues, and furthermore, PCI-32765 that the expression of Nanog was correlated with tumor differentiation and malignancy. These findings also indicate the potential role of Nanog in the diagnosis and prognosis of gastric carcinoma. Cancer stem cells are responsible for the tumorigenicity of tumor cells and lead to tumor recurrence and metastasis (8). Gastric cancer stem cells have the ability to promote the formation PCI-32765 of gastric cancer and maintain the self-renewal and constant proliferation of gastric cancer stem cells (9), suggesting that Nanog may be a new molecular marker for the diagnosis of gastric carcinoma. Previous studies have used qPCR to reveal that the expression levels of Nanog, Sox2, Lin28 and Oct-4 in tumor stem cells were different from other tumor cells and that the performance of miRNA inhibition technology Rabbit polyclonal to C-EBP-beta.The protein encoded by this intronless gene is a bZIP transcription factor which can bind as a homodimer to certain DNA regulatory regions. in the two cell types also varied, suggesting that the two cell types had differing molecular mechanisms (10). Designing a specific miRNA method for cancer stem cells may be more specific and effective than current approaches (10). In the present study, RNA interference (RNAi) technology was used to inhibit the expression of the Nanog gene to study the effect on the tumor biological behavior of the gastric cancer cell line SGC-7901; the aim was to provide an experimental basis for the application of the RNAi technique as a gene therapy method for gastric cancer. Materials and methods Materials The gastric cancer cell line, SGC-7901, strain DH5 and plasmid pGenesil-1 were gifts from Dr Xiang Tingxiu (The First Affiliated Hospital of Chongqing Medical University, Chongqing, China). The annealing buffer contained 10 mM Tris (pH 8.0), 50 Mm NaCl and 1 Mm EDTA dissolved in 50 ml ddH2O, which was then filtered to remove bacteria and stored at 4C. The RNAiso Plus, PrimeScript? RT Reagent kit, Premix Taq? Version 2, restriction endonucleases DH5, then bacteria solution was used to PCI-32765 coat LB solid medium containing kanamycin (25 g/ml), which was incubated at PCI-32765 37C for 16C20 h. Several monoclonal positive colonies were selected the next day and transferred into 4 ml LB liquid medium containing kanamycin (25 g/ml), which was placed in a 37C, 200 rpm shaker to cultivate the bacteria for 12C16 h. E.Z.N.A. Plasmid Mini kit I was used to extract the recombinant plasmid, and enzyme identification and sequencing results demonstrated that the plasmids were correct, indicating that the four recombinant plasmids were constructed effectively. The four recombinant plasmids had been called pshRNA-NanogA, pshRNA-NanogB, pshRNA-negative and pshRNA-NanogC control. Tradition of SGC-7901 cells and transfection A little container of SGC-7901 cell suspension system was eliminated from a ?80C refrigerator, placed in a 37C water bath and constantly agitated gently to ensure rapid thawing. The cell suspension was then transferred to a centrifuge tube, mixed with 2 ml RPMI 1640 medium containing 10% fetal bovine serum and centrifuged for 5 min at 800 g. The supernatant was discarded. The cells were resuspended in 5 ml fresh culture medium containing 10% fetal bovine serum. The cells were transferred into a.