Fig. 4

Ddx3x is the direct target of miR-322. a Quantitative RT–PCR was performed to evaluate the expression of three possible candidate genes using β-actin as the internal control.The relative expression of Ddx3x significantly decreased (1 vs 3, P < 0.05) compared with that of the two other candidate genes, as shown in 4a. b MiR-322 expression was evaluated by real-time PCR after transfection with miR-322 mimics or miRNA mimic NCs using U6 as the internal control. c The relative mRNA expression of Ddx3x after miR-322 inhibition was detected by quantitative RT-PCR using β-actin as the internal control. d, f The relative protein expression of Ddx3x after miR-322 inhibition was determined by Western blot using GAPDH as the internal control.Further analysis revealed that miR-322 expression significantly increased (1 vs 1100; P < 0.05) whereas Ddx3x expression significantly decreased (1 vs 0.43; 1 vs 0.4, P < 0.05) in GC-2 cells transfected with miR-322 mimics (b–d and f). e Sequence alignment of putative amino acids for miR-322 and the 3’UTR of Ddx3x (Ddx3x 3’UTR-WT). The mutant, namely, Ddx3x 3’UTR-MT, is underlined. g MiR-322 directly targeted Ddx3x. Luciferase reporters containing either Ddx3x 3’UTR-WT or Ddx3x 3’UTR-MT were co-transfected with miR-322 mimics or miRNA mimic NCs into GC-2 cells. Reporter activity significantly decreased (1.05 vs 0.58) after miR-322 overexpression compared with the control. All data represent the mean ± SEM of at least three independent experiments (*P < 0.05, ** P < 0.01)