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. 2016 Jul 1;6:28968. doi: 10.1038/srep28968

Figure 4. miR-2861 targets EGFR, AKT2, and CCND1 in cervical cancer cells.

Figure 4

(A) Predicted EGFR, AKT2, and CCND1 3′ UTR binding sites for miR-2861. The alignment of the seed region of miR-2861 with EGFR, AKT2, and CCND1 3′UTR are shown. The mutated sites of targets are indicated in red. (B) EGFR 3′UTR is a target of miR-2861. pmiR-GLO luciferase construct containing a wt (left) or mutated (right) EGFR 3′UTR was cotransfected with miR-2861 or miR-NC in SiHa cells and the luciferase reporter assay was performed at 24 h posttransfection. (C) AKT2 3′UTR is a target of miR-2861. pmiR-GLO luciferase construct containing each wt (wt1, 2, 3, 4, and 5) or mutated (mut1, 2, 3, 4, and 5) AKT2 3′UTR was cotransfected with miR-2861 or miR-NC in SiHa cells, respectively. The luciferase assay was then performed. (D) CCND1 3′UTR is also a target of miR-2861. pmiR-GLO luciferase construct containing a wt (left) or mutated (right) CCND1 3′UTR was cotransfected with miR-2861 or miR-NC and the luciferase assay was performed. (E) miR-2861 overexpression decreased endogenous levels of EGFR, AKT2, and CCND1 proteins in SiHa and CaSki cells. SiHa and CaSki cells were transfected with miR-2861 or miR-NC for 72 h, respectively. EGFR, AKT2, and CCND1 expressions were assessed by Western blot. GAPDH was obtained as a loading control. Error bars, ± SD. *P < 0.05; **P < 0.01; NS, not significant.