Mtor sirna

The normal colon epithelial cell line FHC and human CRC cell lines HT29, HCT116, SW480 and SW620 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and routinely cultured as previously described (17 (link)). All the cell lines used in the present study were authenticated by short tandem repeat (STR) profiling. The GAS5 plasmid for GAS5 overexpression and the hsa-miR-34a mimics or hsa-miR-34a inhibitor were synthesized by GenePharma. The CRC cell lines HT29 and SW480 were transfected with either 200 nM pcDNA3.1-GAS5 or 100 nM miR-34a mimics (or miR-34a inhibitors) for 48 h. The corresponding negative control was performed in all experiments. The mTOR siRNA (50 nM, 48 h) was purchased from Santa Cruz Biotechnology, Inc. Transient transfection of the siRNA or miRNA was conducted using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). A potent mTOR activator, MYH1485 (2 nM, 12 h; Sigma-Aldrich; Merck KGaA), was used to enhance mTOR expression, which in turn inhibited macroautophagy flux. Rapamycin (10 nM, 24 h; Cell Signaling Technology, Inc.), an macroautophagy promoter, was used to further activate CRC cell macroautophagy as previously described (17 (link)).