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Figure 2 | BMC Cancer

Figure 2

From: MicroRNA-302b suppresses cell proliferation by targeting EGFR in human hepatocellular carcinoma SMMC-7721 cells

Figure 2

MiR-302b targets at EGFR. A- miR-302b-binding site at 4259–4284 nt of the EGFR 3′ UTR is predicted to be evolutionarily conserved across different species. B- 24 and 48 h after transfection, luciferase assay in SMMC-7721 cells. 25-bp regions (wt) miR-302b binding sites in the EGFR 3′ UTR was cloned into pmirGLO Dual-Luciferase miRNA Target Expression vector. Identical constructs mutation was generated. Either miR-ctrl or miR-302b was co-transfected with pmirGLO-EGFR-3′ UTR-wt or pmirGLO-EGFR-3′ UTR–mut into SMMC-7721 cells and luciferase activity assayed after 24 h and 48 h. The normalized firefly luciferase activity was obtained by firefly luciferase activity/Renilla luciferase activity. Luciferase activity of reporter gene (EGFR 3′ UTR-wt) displayed a significant decrease by transfecting miR-302b. These experiments were performed in triplicate, and the results are shown as the mean ± s.d. (**P < 0.01 Student’s t-test). C- EGFR mRNA and protein expression levels measured by qRT-PCR and western blot 48 h after transfecting with miR-ctrl, miR-302b, inhibitor-ctrl or miR-302b-inhibitor. The intensity for each band was quantified. The value under each lane indicates the expression level of EGFR, which is represented by the intensity ratio between miR-302b or inhibitor and miR-ctrl or inhibitor-ctrl groups. D- Inverse correlation between miR-302b and EGFR expression in HCC tissues. Expression of EGFR analyzed by qRT–PCR and normalized to β-actin. The miR-302b expression was examined by qRT–PCR analysis and normalized to U6 expression. Statistical analysis was performed using Pearson’s correlation coefficient (r = −0.48, *P < 0.05).

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