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. 2016 Aug 11;12(4):2301–2306. doi: 10.3892/ol.2016.5002

Figure 3.

Figure 3.

miR-186 suppresses CYLD expression by directly targeting the CYLD 3′-UTR and alters the levels of proteins associated with proliferation and cell cycle progression in SKMEL-28 cells. (A) Predicted miR-186c target sequence in the CYLD 3′-UTR and positions of 3 mutated nucleotides (red) in the 3′-UTR of miR-186-mut. (B) CYLD protein expression in SKMEL-28 cells transfected with miR-186 or miR-186-in was detected by western blot analysis. β-actin acted as the loading control. (C) Luciferase reporter assay of SKMEL-28 cells transfected with the pGL3-CYLD-3′-UTR reporter and miR-186, miR-186-in or miR-186-mut with increasing amounts (10 and 50 nM) of oligonucleotides. (D) Quantitative polymerase chain reaction analysis of the expression of cyclin D1 and p21 in SKMEL-28 cells. (E) Western blot analysis of the protein expression of cyclin D1 and p21 in SKMEL-28 cells. β-actin acted as the loading control. *P<0.05 versus respective control. CYLD, cylindromatosis; miR-186, microRNA-186; in, inhibitor; NC, negative control; miR-186-mut, mutated miR-186; 3′-UTR, 3′ untranslated region.