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Figure 4 | BMC Developmental Biology

Figure 4

From: MicroRNA-184 downregulates nuclear receptor corepressor 2 in mouse spermatogenesis

Figure 4

MiR-184 targeted Ncor2 at its 3'UTR. (A) Sequence alignment of the miR-184 complementary site in the 3'-UTRs of Ncor2 mRNAs. The region complementary to the miR-184 seed match highly conserved among human, Macaca mulatta, rat and mouse is in red. And the overlapping complementary region in rat and mouse to the miR-184 seed match is in red or boxed. (B) Constructs and mutated target sequence of Ncor2. A 517 bp region of the 3'-UTR of mouse Ncor2 containing three potential miR-184 target sites was cloned into the firefly luciferase vector (Ncor2-wt). For Ncor2-mut, six nt (underlined and in yellow) of the overlapping complementary sites were mutated. (C) Quantitative RT-PCR showed the increased levels of miR-184 upon overexpression. (D) Dual-luciferase reporter assay. Ncor2-wt construct in the presence of 10 pmol of miR-184 mimic showed the inhibitory activity of this reporter. The Ncor2-mut construct showed that miR-184 mimic cannot inhibit the luciferase activity compared with the wild-type construct. This experiment was repeated three times in triplicate. Values are presented as the mean ± SD. ***P < 0.001. (E) Western-blot assay. NCOR2 protein levels decreased in Hela cells after overexpression of miR-184 mimic (lane 184-mim) for 48 h compared with the cells transfected with scrambled control oligonucleotide (lane scramble). Arabic numbers demonstrate the NCOR2 average gray value normalized to β-Actin average gray value. (F) Quantitative RT-PCR showed Ncor2 mRNA did not change after overexpression of miR-184 in Hela cells compared with the negative control.

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