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. 2014 Apr 14;3(4):e97. doi: 10.1038/oncsis.2014.11

Figure 3.

Figure 3

Ago2 is directly targeted by miR-99a. (a) Schematic representation of the 3′-UTR of Ago2. Red bars show putative miR-99a target sequences. Sequence alignment of the target site among vertebrate, including amphibians (Xenopus (Xtr)), birds (Gallus (Gga)) and mammals (human (Hsa) and mouse (Mmu)), is shown below. (b, c) The capacity of miR-99a on regulating Ago2 as detected by western blot and qRT–PCR. Ad-blank, the control adenovirus without transgene; Ad5-miR-99a, the recombinant adenovirus expressing miR-99a; Ctrl, untreated HCC cells. (d) Luciferase assay for the regulation capacity of miR-99a on Ago2. Both wild and mutated (mut) Ago2 3′-UTR (the putative seed binding sequence was mutated from TACGGGTC to ATGCCCAG) was used. Empty, Huh7 cells transfected with the empty dual-Luc assay vector, psiCHECK-2; Ctrl, Huh7 cells transfected with psiCHECK-2 containing Ago2 3′-UTR (psiCHECK2-Ago2 UTR) or mutated Ago2 3′-UTR (psiCHECK2-Ago2 mutUTR), respectively. miR-99a, Huh7 cells co-transfected with miR-99a mimic and psiCHECK2-Ago2 UTR/psiCHECK2-Ago2 mutUTR; inhibitor, Huh7 cells co-transfected with miR-99a inhibitor, miR-99a mimic and psiCHECK2-Ago2 UTR/psiCHECK2-Ago2 mutUTR. *P<0.05; **P<0.01. (e) Overexpression of AGO2 protein by transduction with Ad5-Ago2 (MOI=5), a recombinant adenoviral vector expressing Ago2. (f) The counteraction of AGO2 overexpression on miR-99a killing effect on HCC cells as examined by MTT assay.