Mir 497 mimics

A series of CRC cell lines (SW480, SW620, HT29, HCT116) and human normal colon epithelial cell (FHC), obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin in a humidified incubator (37°C, 5% CO₂).
miR-497 mimics and negative mimics control (NC) were purchased from GenePharma Co., Ltd. (Shanghai, China). To overexpress TTN-AS1, the full-length sequence of TTN-AS1 cDNA was cloned into the pcDNA3.1 vector (Invitrogen). An empty pcDNA3.1 vector was used as a negative control. To knockdown TTN-AS1, chemically synthesized siRNA sequence targeting TTN-AS1 was inserted into the shRNA expression vector pGPH1/Neo (GenePharma Co., Ltd.). Cells were cultured to about 80% confluence and then transfected with the vectors or oligonucleotides using Lipofectamine 2000 (Invitrogen). After 48 hrs, the transfection efficacy was verified by RT-qPCR analysis. In some cases, cells were treated with a specific Akt activator, SC79 (5 μM; Invitrogen).7 (link)

Cells in FBS-free DMEM were seeded into six-well plates at a density of 60–70% confluence. After adherence, cells were transfected with miR-497 mimics, negative control miRNA mimics (miR-NC), AKT3 small interfering (si)RNA, NC siRNA, pCDNA3.1-AKT3 or pCDNA3.1 blank vector using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.). After 6–8 h of incubation, culture medium was replaced by DMEM supplemented with 10% FBS. miR-497 mimics and miR-NC were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). AKT3 siRNA or NC siRNA were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). pCDNA3.1-AKT3 and a pCDNA 3.1 blank vector were synthesized by the Chinese Academy of Sciences (Changchun, China).