Fig. 2

Luciferase and CHIP assay. (A) The luciferase experiment was used to evaluate the binding sites of hsa-miR19-3p to 3’-UTR of NKRF. Upper, prediction of binding sites of hsa-miR19-3p to 3’-UTR of NKRF. Down, analysis of differences between groups. The histogram shows the relative activity of luciferase. *, p < 0.05, and **, p < 0.01. (B) The luciferase experiment was used to evaluate whether ICL can weaken the binds of hsa-miR-19-3p to NKRF 3’-UTR. Upper, prediction of binding sites of hsa-miR19-3p to ICL. Down, analysis of differences between groups. (C) Evaluation of the activity of hsa-miR19-3p promoter. 48 h after transfection, we can evaluate the promoter activity by observing the expression of GFP in 293T cells. (D)The luciferase experiment was used to evaluate whether there is a TFBS of NF-κB in the hsa-miR-19-3p promoter. TNF-α (5ng/mL) was used to activate NF-κB in 293T cells. If NF-κB could bind to miR-19-3p promoter, the addition of TNF-α would inevitably up-regulate the luciferase activity in 293T cells 48 h after transfection. (E) CHIP-PCR was used to verify the TFBS of NF-κB locus on the promoters of hsa-miR19-3p. RT-PCR was used to detect whether PCR amplification with different eluents as templates had the single product. Five microliters PCR products was taking for 2% agarose electrophoresis. β-actin was used as reference. The tests were carried out on three biological triplicates, and data are expressed as the mean ± SD. ** p < 0.01, * p < 0.05