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. 2017 Jan 18;8(9):14693–14707. doi: 10.18632/oncotarget.14711

Figure 4. MiR-744 activates Wnt/β-catenin pathway by targeting multiple negative regulators and NKD1 is a crucial direct target of miR-744.

Figure 4

(A) Top ten pathways sort by Kegg and Biocarta enrichment pathways result from array analysis. (B) Transfected with TOPflash or FOPflash and Renilla pRL-TK plasmids were subjected to dual-luciferase assays in PC3 and DU145 cells. Reporter activity detected was normalized by Renilla luciferase activity at 48h post-transfection. (C) Luciferase activity of TOP flash/FOP flash in the indicated cells. Each bar represents the mean ± SD of three independent experiments. *P < 0.05. (D) Protein expression of NKD1, GSK3β, SFRP1, TLE3 and nuclear β-catenin in both PC3 and DU145 cells was determined by Western blot assay after anti-mir-744 or anti-NC transfection. GAPDH was used as an internal control as well as P84 was served as an internal control in nucleus. (E) IHC staining with an anti-NKD1 antibody on PC3 xenograft tumors, we found that NKD1 positive cells in LV-anti-miR-744 treated PC3 xenograft tumors were much more than in the negative control tumors. (F) ISH and IHC staining on ADPC tissues and CRPC tissues with a miR-744 probe and an anti-NKD1 antibody, we observed that NKD1 expression was inversely correlated with miR-744 level. Scale bars represent 50 μm and 100 μm. (G) Overlap of miRNA target bioinformatic prediction methods, inhibition of mir-744 induced upregulated mRNAs and the negative regulators of Wnt signaling pathway. NKD1 was common to the three lists. (H) Principle scheme with RNA sequence alignment presented that the 3′-UTR of NKD1 mRNA contained a complementary site for the seed region of miR-744 (5138–5161bp) predicted by RNA22-HSA. I. The luciferase activity was detected that psiCHECK-2 luciferase reporter vector containing wild type and mutations of the binding sites in the 3′UTR of NKD1 mRNA with the miR-744 minics or miR-NC were co-transfected into PC3 and DU145 cells for 48 h. NKD1 mut was replaced the complementary region by a mutant as negative control. Rluc activity in the cells was measured and normalized to Fluc activity. Each bar represents the mean ± SD of three independent experiments. *P < 0.05.