Pnf κb luc vector

For luciferase assay, U251 or LN229 cells were seeded into 24-well plates. After culturing for 24 hours, pNF-κB-luc vector (Beyotime) and Renilla vector (pRL-TK; Promega Corporation, Fitchburg, WI, USA) with or without H19 knockdown or overexpression were co-transfected into glioma cells using Lipofectamine^®2000. Luciferase activity levels were measured 48 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized using the corresponding Renilla luciferase activity. Data are representative of three independent experiments.

For the miR‐192‐target analysis, two fragments of NKRF‐WT or NKRF‐Mut were cloned into the downstream of the pMIR‐GLO dual luciferase miRNA target vector according to protocol. pNFκB‐luc vector was purchased from Beyotime which contained several κB sites reflecting the transcriptional activity of NF‐κB. In a 48‐well plate format, a final concentration of 1 μg/ml of reporter plasmid DNA and 100 nmol/l of miR‐192 mimics or 100 nmol/l of miR‐192 inhibitor mixed in 250 μl of serum‐free medium were used for each transfection. In the cotransfection system, 50 ng pRL‐SV40 reporter plasmids, 100 nmol/l of miR‐192 mimics or 200 nmol/l of miR‐192 inhibitor and 250 ng reporter plasmid DNA were added. At 48 h post‐transfection, luciferase activities using the dual‐luciferase assay system were measured (Promega).