Overexpression of miR 425 was enough to downregulate PTEN expression at each the protein and mRNA levels in AGS cells. Accordingly, IL 1B induced PTEN repression was rescued by expressing anti miR 425 in AGS cells. Anti miR 425 was able to up regulate PTEN expression in NCI N87 cells with no IL 1B stimulation. Our data also indicated that the 3 UTR is essential for miR 425 mediated PTEN downregulation due to the fact expression of a PTEN coding area construct was insensitive to miR 425 overexpression and IL 1B induction in AGS cells. Taken collectively, these final results indicate that miR 425 plays a important role in repressing PTEN expression by targeting its three UTR upon IL 1B induction.
IL 1B induced NF kappaB activation is essential for miR 425 induction To decide the mechanism involved in miR 425 trans activation upon IL 1B induction, we examined the im pact of different kinase inhibitors on miR 425 induction mtorc2 inhibitor in IL 1B treated AGS cells. IL 1B induced miR 425 up regulation was drastically inhibited by the IKK inhibi tor TPCA 1 but not by the p38 MAPK inhibitor BIX02188 or the JNK inhibitor SP600125. Previous studies have demonstrated that IKK is definitely an es sential kinase needed for NF kappaB signaling, there fore, this outcome indicated the crucial part of NF kappaB signaling inside the regulation of miR 425 transcription upon IL 1B induction. Regularly, induction of pri miR 425 upon IL 1B remedy was remarkably inhibited within the presence of your IKK inhibitor or siRNAs for NF kappaB. We also observed that IL 1B induced PTEN repression was attenuated inside the presence from the IKK in hibitor or siRNAs for NF kappaB.
To deter mine irrespective of whether NF kappaB activity was present selleck in AGS cells treated with IL 1B, we utilized a western blot to deter mine the level of phosphorylated NF kappaB p65. The level of phosphorylated NF kappaB p65 was high in AGS cells treated with IL 1B. Moreover, silencing of NF kappaB inhibited miR 425 expression in NCI N87 cells devoid of IL 1B treatment. These results suggesting that IKK dependent NF kappaB activation upon IL 1B treat ment is necessary for PTEN downregulation, probably by way of its enhancement of miR 425 transcription. To determine regardless of whether NF kappaB straight regulates miR 425 transcription, we analyzed the upstream se quences of miR 425 using the WeightMatrix library and identified three possible NF kappaB binding web sites in the promoter area of miR 425. We performed chromatin immunoprecipita tion assays with AGS cancer cells applying monoclo nal anti NF kappaB antibodies. As shown in Figure 4B, only primer B of miR 425 developed strong PCR merchandise, which recommended that the NF kappaB protein formed com plexes with all the B binding website inside the miR 425 promoter.