Figure 1.

Scheme of putative microRNA-18a (miR-18a) binding sites in the Dicer Ⅰ mRNA 3’-untranslated region (3’-UTR). Two putative binding sites for miR-18a are present. Mutations were generated in the Dicer Ⅰ 3′-UTR sequence in the complementary sites for the miR-18a seed regions."

Figure 2.

Reducing miR-18a expression degrades migration and invasion of HCC cells. Transwell migration assays of HepG2 cells (A) or HepG2.2.15 cells (B) expressing miR-18a inhibitor or inhibitor negative control (inhibitor NC). Transwell matrigel invasion assay of HepG2 cells (C) or HepG2.2.15 cells (D) expressing miR-18a inhibitor or inhibitor NC. Representative light microscopy images (×200 magnification) are shown on the left, and the histogram showing the migrated cell number is shown on the right. The values shown are expressed as mean±SD.*P<0.01 compared with the inhibitor NC (n=3)."

Figure 3.

Wound healing assays of HepG2 cells (A) and HepG2.2.15 cells (B) after treatment with miR-18a inhibitors or inhibitor NC. The wound healing rates of wound closure per field is shown.*P<0.01 compared with the inhibitor NC (n=3)."

Figure 4.

miR-18a post-transcriptionally downregulates Dicer Ⅰ expression by directly targeting the Dicer Ⅰ mRNA 3′-UTR. A human Dicer Ⅰ 3′-UTR fragment containing either the wild-type (WT) or mutant (Mut) miR-18a-binding sequences was cloned downstream of the luciferase reporter gene. Luciferase assays verified direct binding of miR-18a to the predicted sites. HEK293 cells were co-transfected with the luciferase construct and miR-18a mimics or control miRNA. The Renilla construct was also co-transfected as an internal control. Luciferase activity was normalized to Renilla luciferase activity. The data are presented as the mean ± SD of two experiments with four replicates each.*P<0.01 compared with the mimic NC (n=4)."

Figure 5.

Endogenous Dicer Ⅰ expression levels were detected in HepG2 and HepG2.2.15 cells transfected with miRNA-18a inhibitor or Dicer Ⅰ small interfering RNA (siRNA) for 48 hours by real-time RT-PCR (A) and western blotting (B). a. inhibitor NC; b. miRNA-18a inhibitor; c. miRNA-18a inhibitor + si-RNA NC; d. miRNA-18a inhibitor + si-Dicer Ⅰ.The data represent the mean ± SD of three independent experiments.*P<0.01 compared with the inhibitor NC (n=3); #P<0.01 compared with the miRNA-18a inhibitor + si-RNA NC (n=3)."

Figure 6.

Downregulation of Dicer Ⅰ promotes cell migration and invasion in HCC cells. The effects of miRNA-18a inhibitor or restoration of miRNA-18a function through downregulation of Dicer Ⅰ on cell migration and invasion were confirmed by transwell migration and invasion assays in HepG2 (A and C) and HepG2.2.15 (B and D) cells transfected with miRNA-18a inhibitor or si-Dicer Ⅰ. The data represent the mean ± SD of three independent experiments.*P<0.01 compared with the inhibitor NC (n=3); #P<0.01 compared with the miRNA-18a inhibitor + si-RNA NC (n=3)."

Figure 7.

Wound healing assays of HepG2 cells (A) and HepG2.2.15 cells (B) treated with miRNA-18a inhibitor or si-Dicer Ⅰ. The wound healing rate of wound closure per field is shown. #P<0.05, *P<0.01 compared with the miRNA-18a inhibitor + siRNA NC (n=3)."