Figure 1. miR-20a-5p negatively regulated Smad4 in human colorectal cancer.

(A) 293T cells were co-transfected with the negative control (NC) or miRNA mimics and the luciferase reporter construct containing the wild-type Smad4 3′-UTR. The luciferase activity of the NC was set to 1, the others were normalized to the NC. *P < 0.05 was considered to be statistically significant. (B and C) The mRNA expression of miR-20a-5p (P = 0.0002) and Smad4 (P < 0.001) in 10 CRC patients’ tissues with and without metastasis was evaluated by qRT-PCR. (D) The correlation between miR-20a-5p and Smad4 mRNA expressions in 10 colorectal cancer patients was evaluated using Spearman's correlation analysis (P = 0.031, R² = 0.234). (E) The expression of miR-20a-5p in different colorectal cancer cells was evaluated by qRT-PCR. FHC was used as a control. (F) HT29 cells were infected with miR-20a-5p-expressing lentiviral vector and HCT116 cells were infected with miR-20a-5p-knockingdown lentiviral vector. The luciferase reporter construct containing the wild-type Smad4 3′-UTR. Empty vector was used as a control, *P < 0.05. (G) The expression levels of Smad4 mRNA and protein were evaluated using qRT–PCR and western blot analysis in empty vector-infected or miR-20a-5p vector infected HT29 cells and negative control or anti-miR-20a-5p vector infected HCT116 cells, respectively, *P < 0.05.