Figure_5.tif (1.02 MB)

Effects of miR-21 knockdown and Pten down-regulation in Pc 9/GR cell lines.

figure

posted on 2014-07-24, 03:04 authored by Hua Shen, Fang Zhu, Jinyuan Liu, Tongpeng Xu, Dong Pei, Rong Wang, Yingying Qian, Qi Li, Lin Wang, Zhumei Shi, Jitai Zheng, Qiudan Chen, Binghua Jiang, Yongqian Shu

(A) Relative expression levels of miR-21 in pc-9 GR/inhibitor NC+scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were assessed by stem-loop qRT-PCR, and normalized to the levels of U6. Triplicate assays were performed for each RNA sample. Significant differences are indicated by **(P<0.01). (B) Gefitinib sensitivity of pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were evaluated by CCK-8 assay. (C) IC50 values for gefitinib in PC9 GR/21 inhibitor+scramble siRNA(siSCR) transfected cells were significantly lower than in PC9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells(**p<0.01) and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells(#p<0.05). (D) pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were used for cell invasion assays. Invasion cells stained by 0.1% crystal violet were counted 24 h after plating. Upper panels: representative photographs of invasive cells (×200). (E) Number of invasive cells per field was obtained from at least three replicate wells and represented by mean ± SD (bottom graph). **indicates significant difference between pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells (P<0.01). ## indicates significant difference between pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells (P<0.01). (F) Apoptosis rate of pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells induced by gefitinib were evaluated by flow cytometry. All cells were treated with 1 µmol/L gefitinib for 72 hours before apoptosis evaluation. (G) Pten, p-AKT, AKT, p-ERK, and ERK proteins in pc-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells, pc-9 GR/miR-21 inhibitor+scramble siRNA(siSCR) transfected cells and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells were analyzed by Western blot. (H) Quantification of western blot showed up-regulated expression of Pten protein in PC-9 GR/21 inhibitor+scramble siRNA(siSCR) transfected cells compared with PC-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells (*P<0.05) and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells(#P<0.05); p-AKT and p-ERK proteins expression was down-regulated in PC-9 GR/21 inhibitor+scramble siRNA(siSCR) transfected cells compared with PC-9 GR/inhibitor NC+ scramble siRNA(siSCR) transfected cells (*P<0.05) and pc-9 GR/miR-21 inhibitor+Pten siRNA transfected cells (#P<0.05). There were no significant differences in total AKT and total ERK proteins among the three cells.