SATB1-shRNA). Discussion NPC is one of the common malignancies of epithelial origin in head and neck cancers 2, 28. of cell proliferation and invasion were assessed using MTT and transwell assays, respectively. Drug and radiation resistance assays were performed after SATB1 knockdown and cell viability was detected by MTT method. Results: SATB1, MMP-9 and Vimentin were markedly upregulated in 5-8F/DDP and 5-8F/R cells compared with 5-8F cell, whereas E-cadherin was obviously downregulated. 5-8F/DDP and 5-8F/R cells displayed drug and radiation resistance to DDP or X-irradiation, respectively, while DDP or X-irradiation inhibited 5-8F cell viability in a time- and dose-dependent manner. Subsequently, knockdown of SATB1 resulted in decreased MMP-9 and Vimentin expression and increased E-cadherin expression in 5-8F/DDP and 5-8F/R. Furthermore, silencing of SATB1 suppressed proliferative and invasive abilities of 5-8F/DDP and 5-8F/R cells. Additionally, SATB1 knockdown reduced drug resistance of 5-8F/DDP cell to DDP and decreased radiation resistance of 5-8F/R cell to X-ray. Conclusion: These results suggest that high expression of SATB1 plays an important role in the malignant behavior of NPC and leads to X-radiation and drug resistance in NPC through promoting EMT process and enhancing MMP-9 expression. SATB1 may be a promising therapeutic target for aggressive and chemoradiation resistant NPC. At the different time points, cells were treated with DDP at different concentration (0.5, 1, 2 and 4 g/ml) or X-irradiation at different dose (2, 4, 6 and 8 Gy), respectively. After treatment, the cells were incubated with 20 l 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution reagent (Sigma-Aldrich Corp.) for 4 h, and then the cell viability was evaluated by MTT assay. The absorbance at 490 nm (A490) was measured using a microplate reader (Bio-Tek Inc., Winooski, VT, USA)cells were dispensed within 96-well plates at a density of 5 103 per well and cultured Oxypurinol at 37C in 5% CO2After 16 h, the cells were treated with 10 l 1 g/ml DDP. After 48 h incubation, cells were treated with 20 l MTT (5 mg/ml) solution reagent (Sigma-Aldrich Corp.) for 4 h. The cell viability was assessed by MTT assay. A490 was measured using a microplate reader (Bio-Tek Inc.). The cell growth inhibition rate indirectly indicates the drug resistance ability of 5-8F/DDP cell to DDP. Radiation resistance assay after RNAi After 5-8F/R cell was successfully transfected, each group (SATB1-shRNA, NC-shRNA and WT) cells were dispensed within 96-well plates at a density of 5 103 per well and cultured at 37C in 5% CO2 for 16 hThe cells were exposed to 4 Gy of X-ray for 48h and then cells were treated Rabbit Polyclonal to MEF2C with 20 l MTT (5 mg/ml) solution reagent (Sigma-Aldrich Oxypurinol Corp.) for 4 h. Cell viability was measured by MTT assay. A490 was assayed using a microplate reader (Bio-Tek Inc.). The cell growth inhibition rate indirectly indicates the radiation resistance ability Oxypurinol of 5-8F/R cell to X-irradiation. Statistical analysis All measurement experiments were repeated three times independently, and the data are presented as mean standard deviation (SD). Comparisons between two groups were performed using Student’s t-test. Comparisons among multiple groups were performed using ANOVA. Statistical significance was defined as a P-value<0.05. Results Chemoradiation resistance of 5-8F/DDP and 5-8F/R prior to SATB1 knockdown MTT cell viability assay was used to determine the chemoradiation resistance of 5-8F/DDP and 5-8F/R. The inhibitory effects of DDP on 5-8F and 5-8F/DDP cells were investigated by varying incubation times at drug concentrations of 0.5, 1, 2 and 4 g/ml. The inhibitory effects of X-ray on 5-8F and 5-8F/R cells were investigated by varying incubation times at radiation doses of 2, 4, 6 and 8 Gy. As illustrated in Figure ?Figure11A and 1B, the 5-8F cells showed gradually reduced cell viability when treated with serial increased dose of DDP and X-ray or exposed to the indicated dose at serial increased times. Thus, DDP and X-irradiation caused an.