Figure 3. SOCS1 is the direct target of miR-30d.

(A) Bioinformatics analysis performed using the prediction algorithms provided by TargetScan, PicTar, and miRanda. Each labeled circle represents 1 prediction algorithm and the number of predicted genes; the number listed in the overlapping circles indicates the number of genes that were simultaneously predicted by the different algorithms. (B) qPCR analysis of the expression levels of the target genes. The upregulated mRNA levels confirm each gene's expression compared with the control, cells transfected with anti-miR-30d, and control oligonucleotides in the PCa cell lines. Red and blue indicate high and low levels of mRNA expression, respectively. (C) The miR-30d transfection efficiency HEK293T cells were confirmed using qPCR. (D) Reporter assay in HEK293T cells transiently cotransfected with the each reporter construct, and miR-30d was compared with the control cells. All experiments were repeated 3 times. Values represent the means, and the error bar represents the SD. **P < 0.01. (E) The transfection efficiency of miR-30d in PC3 and LNCaP was confirmed using qPCR (RWPE-1-30d, PC3-30d and LNCaP-30d). (F) Identification of the miR-30d-binding region in the WT SOCS1 3'-UTR and mutated sequences. (G) Relative luciferase activities were analyzed, and the stable expression of miR-30d in prostate cell lines that had been transfected with the WT or Mut 3'-UTR reporter constructs were compared with the control cells. All experiments were repeated 3 times. Values represent the means, and the error bar represents the SD. *P < 0.05 and **P < 0.01 according to Analysis of variance (ANOVA) followed by the Tukey-Kramer test. (H) Western blot assays of the endogenous SOCS1 protein levels in the prostate cell lines. (I) SOCS1 protein levels were analyzed by Western blot using PC3 and LNCaP cells that had been transiently transfected with 40 nM anti-miR-30d or control oligonucleotides. N.D, not detected.