Figure 4

Dicer-1 is direct target of miR-107.

(A) Heat-map of miRNAs that were differentially expressed at least 1.5-fold between RBMECs transfected with miR-107 and RBMECs transfected with scramble probe. (B)The putative targets genes of miR-107. (C) The expression of Dicer-1 in HUVECs, RBMECs and astrocytes under OGD for 12 h. *P < 0.05, vs. control. (D) The mRNA levels of Dicer-1 in rat IBZ on 3rd and 7th days after pMCAO were detected by qRT-PCR. *P < 0.05, vs. control. (E) Representative picture and analysis diagram show the protein levels of Dicer-1 in HUVECs as determined by Western blotting. *P < 0.05, vs. Control, ^ΔP < 0.05, vs. sham group. (F,G) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. (H,I) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with anti-miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. *P < 0.05, vs. control. (J) After pMCAO, rats were injected with antagomir control (antagomir-ctl) or antagomir-107 and divided into 3 group: pMCAO (control), negative control (antagomir-ctl) and antagomir-107. The mRNA level of Dicer-1 in the three groups were detected by using qRT-PCR on 7th day after pMCAO. *P < 0.05, vs. control, ^#P < 0.05, vs. antagomir-ctl group. (K) Luciferase activity of reporter constructs carrying luciferase cDNA and parts of the 3′UTR of target mRNAs. Left, Localization of binding sites for human miR-107 (hsa-miR-107) in the 3′UTR of target mRNA and their evolutionary conservation. Right, Quantitative analysis of 3′UTR luciferase activities. HUVECs were transfected with miR-107 or anti-miR-107 (in parallel with control molecules miR-ctl and anti-miR-ctl). Data are from 3 independent experiments performed in triplicate. Data are presented as mean ± SD. *P < 0.05, vs. control.