Immortalized Cell Lines for Functional Analysis

HaCaT (ATCC, Manassas, VA), a spontaneously transformed immortal keratinocyte cell line from skin was maintained in high glucose DMEM medium (Gibco) supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen), and TIGK (gift from Dr. Richard J. Lamont, University of Louisville), a telomerase immortalized epithelial cell line from oral mucosa (gingiva) was grown in DermaLife K medium (Lifeline Cell Technology) supplemented Life Factors (Lifeline Cell Technology), as previously described^{27 (link),44 (link)}. For functional analysis, miR-21 mimic and a control non-targeting miRNA mimic (GE Healthcare Dharmacon, Lafayette, CO), or anti-miR-10b LNA inhibitor and negative control LNA (QIAGEN, Hilden, Germany), were transfected into cells using DharmaFECT Transfection Reagent 1 as described previously^{45 (link)–47 (link)}. Cell proliferation was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay as described previously^{26 (link),48 (link)}. In vitro cell migration was measured using the Oris 96-well cell migration assay kit (Platypus Technologies, Fitchburg, WI) as described previously^{45 (link)}. The cells that migrated into the detection zone were counted using ImageJ analysis software (version 1.421).