MiR 362 targeted CYLD directly CYLD deubiquitinase is a key negative regulator of NF B signaling. Analysis using publicly avail able algorithms showed that CYLD is a potential target of miR 362. Western blotting Pazopanib analysis revealed that CYLD expression was dramatically repressed by miR 362 overexpression, or induced by miR 362 inhibition. To examine whether miR 362 induced CYLD downregulation was mediated by the CYLD 3 UTR, we subcloned the CYLD 3 UTR fragment containing the miR 362 binding site into pEGFP C1 and pGL3 dual luciferase reporter vectors. Inhibitors,Modulators,Libraries MiR 362 overexpression only decreased the expression of the GFP vector containing the CYLD 3 UTR, but had no effect on GFP tubulin expression, suggest ing that miR 362 specifically affected the CYLD 3 UTR.

Reduced luciferase activity was observed following miR 362 overexpression in both BGC 823 and SGC 7901 cells, whereas the repressive effect of miR 362 on luciferase ac tivity of Inhibitors,Modulators,Libraries the CYLD 3 UTR was abolished by the miR 362 inhibitor. MiR 362 overexpression had no ef fect on the luciferase Inhibitors,Modulators,Libraries activity of CYLD 3 UTR mut, which contained point mutations Inhibitors,Modulators,Libraries in the miR 362 binding seed region of the CYLD 3 UTR. Collectively, our results demonstrate that CYLD is a bona fide target of miR 362. CYLD downregulation is critical for miR 362 mediated NF B activation To further investigate the role of CYLD repression in miR 362 mediated NF B activation, we examined the effects of CYLD downregulation on NF B activation in BGC 823 and SGC 7901 cells. As expected, CYLD knockdown by the two CYLD specific siRNAs signifi cantly increased NF B reporter luciferase activity and the expression levels of the eight NF B target genes.

However, further miR 362 overexpression in the CYLD silenced cells did not have a significant additive effect on NF B reporter luciferase activity nor NF B Inhibitors,Modulators,Libraries target genes ex pression. Importantly, CYLD downregulation abolished the miR 362 inhibition that induced repression of NF B activity and target gene expression. Overall, our results demonstrate that CYLD plays an important role in miR 362 mediated NF B activation. Clinical correlation between miR 362, CYLD expression, and NF B activation in gastric cancer tissues We investigated whether the miR 362 induced CYLD repression and NF B activation were clinically relevant.

MiR 362 levels in the 10 freshly collected obviously gastric cancer specimens were inversely correlated with CYLD expression levels but positively corre lated with nuclear p65 expression. Altogether, our results suggest that miR 362 upregulation activates NF B signaling by repressing CYLD, conse quently leading to cell proliferation and apoptosis resist ance in gastric cancer. Discussion MiRNAs are small noncoding RNAs that regulate the ex pression of a large number of intracellular target genes.