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. 2020 May 12;10:589. doi: 10.3389/fonc.2020.00589

Figure 2.

Figure 2

TP53INP1 is the direct target of miR-221. (A) The protein expression of p62, LC3, TP53INP1, p-ERK1/2, and ERK1/2 in T24 cells treated by miR-221 inhibitor and 5637 cells treated by miR-221 mimic. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. (B) Schematic illustration of the sequence of hsa-miR-221 and its complementary sequence in 3′UTR of TP53INP1 mRNA, in which the letters in red represent the five nucleotides we mutated. Luciferase activity assays using luciferase reporters with wild-type TP53INP1 3′UTR (TP53INP1 WT) or mutant TP53INP1 3′UTR (TP53INP1 MUT) were performed with co-transfection of miR-221 mimics or negative control into 5637 bladder cancer (BC) cells and 293T cells. Data represent the mean ± SD of triplicates. (C–E) T24 cells were transfected with miR-221 inhibitor or Lt-TP53INP1 for 48 h. A portion of the cells were harvested for IB analysis (C). The other portion of cells were seeded in the upper chamber of the transwell system (D) or plated into six-well plates for 24 h, followed by wound healing assay (E). Scale bar, 50 μm (D), 100 μm (E). *p < 0.05, **p < 0.01, and ***p < 0.001.