2013;14:215C223. to evaluate the effect of miR-216b mimics (50 pmol/ml) or inhibitors (50 pmol/ml) on changing IC50 of cisplatin to A549 cells. *NCO group. (D) Effect of miR-216b mimics (50 pmol/ml) or inhibitors (50 pmol/ml) on changing IC50 of cisplatin to PC9 cells. *NCO group. MiR-216b targets c-Jun in NSCLC To explore the mechanism by which miR-216b sensitized NSCLC cells to cisplatin, TargetScan, miRanda, and PicTar public databases were used to predict the potential target of miR-216b in NSCLC. We observed that this oncogene of c-Jun made up of putative binding sequence paired with miR-216b at the 3 UTR of its mRNA (Physique ?(Figure2A).2A). To confirm that miR-216b targets c-Jun in NSCLC, luciferase reporter assays were performed. The results showed that co-transfection with miR-216b mimics significantly decreased the luciferase activities of pMIR reporters made up of wild type (WT) c-Jun 3 UTR in both A549 and PC9 NSCLC cells. However, miR-216b exhibited no effect on the pMIR reporters made up of mutant type (MT) c-Jun 3 UTR (Physique ?(Figure2B).2B). We thus exhibited that miR-216b targets c-Jun in NSCLC. To test the effect of miR-216b on cisplatin-induced upregulation of c-Jun, we detected the protein level of c-Jun in NSCLC cell lines after they were treated with cisplatin and miR-216b. As shown in Physique ?Physique2C,2C, single treatment of miR-216b was able to decrease the expression of c-Jun in A549 and PC9 NSCLC cells. Moreover, transfection with miR-216b was found to abolish the upregulation of c-Jun induced by cisplatin. These data indicated that miR-216b suppressed the overexpression of c-Jun in cisplatin-treated NSCLC cells. Open in a separate window Physique 2 MiR-216b suppresses c-Jun expression in NSCLC(A) Putative binding sequence of c-Jun mRNA paired with miR-216b. (B) After co-transfection with miR-216b (50 pmol/ml) and pMIR reporters (2 g/ml) in A549 and PC9 NSCLC cells, relative luciferase activities of pMIR reporters were measured by using Dual-Luciferase Reporter System. *NCO group. (C) Effect of miR-216b (50 pmol/ml) and cisplatin (2 M) on changing protein level of c-Jun in A549 and PC9 NSCLC cells. MiR-216b sensitizes NSCLC cells to cisplatin treatment through decreasing the expression of c-Jun As c-Jun was targeted by miR-216b, we were supposed to explore whether the miR-216b-sensitized cell death in cisplatin-treated NSCLC cells was dependent on the suppression of c-Jun. We therefore overexpressed the c-Jun in A549 and Personal computer9 NSCLC cells by transfection with recombinant manifestation vector of c-Jun (Shape ?(Figure3A).3A). Although miR-216b improved the cytotoxicity of cisplatin to NSCLC cells significantly, enforced manifestation of c-Jun considerably inhibited the synergistic aftereffect of miR-216b (Shape ?(Figure3B).3B). Furthermore, we noticed that miR-216b considerably enhanced the power of cisplatin to induce apoptosis of NSCLC cells. Nevertheless, restore of c-Jun avoided the miR-216b-advertised apoptosis when the NSCLC cells had been beneath the cisplatin treatment (Shape ?(Shape3C).3C). These outcomes indicated how the miR-216b-sensitized apoptotic Rabbit Polyclonal to UBTD2 cell loss of life in cisplatin-treated NSCLC cells was reliant on the suppression of c-Jun. Next, we knockdown the expression of c-Jun in NSCLC cells by transfection using its particular siRNA directly. We noticed that the result of c-Jun siRNA was identical with miR-216b. C-Jun siRNA treatment can also sensitize NSCLC cells to cisplatin-induced cytotoxicity (Shape ?(Figure3D).3D). We Dopamine hydrochloride emphasized the need Dopamine hydrochloride for c-Jun suppression in miR-216b-promoted cell loss of life Dopamine hydrochloride therefore. Open in another window Shape 3 MiR-216b sensitizes NSCLC cells to cisplatin treatment through reducing the manifestation of c-Jun(A) European blot evaluation was performed to judge the result of c-Jun siRNA (50 pmol/ml) and c-Jun plasmid (2 g/ml) on changing the mobile proteins degree of c-Jun in A549 and.
