(A) The bindng site of miR-891b to Cbl-b 3′UTR and construction of plasmids containing Cbl-b 3′UTR sequence or binding site-mutated sequence. (B) miR-891b inhibited the expression of Cbl-b at the post-transcriptional level. PDAC cell lines, SW1990 and PANC-1, were transfected with miR-891b mimic in different concentrations. Western blot indicated miR-891b down-regulated the expression of Cbl-b protein. RT-PCR suggested overexpression of miR-891b did not significantly affect the level of Cbl-b mRNA. (C) The pancreatic cancer cell line, Capan-2, and the human colon cancer cell lines, HCT 116 and RKO, as well as the human gastric cancer cell lines, SGC-7901 and MGC-803, were transfected with miR-891b mimic or NC. Western blot showed that overexpression of miR-891b significantly down-regulated the level of Cbl-b protein in the above cell lines. (D) The PDAC cell lines, SW1990 and PANC-1, were co-transfected with pMirTarget-Cbl-b WT (or MUT) plasmid, miR-891b mimic (or NC), and pRL-TK. The dual luciferase reporter assay detected the activity of luciferase. The histogram shows the relative activity of luciferase (mean ± SD, results of three independent experiments, *P < 0.05). The results suggest that miR-891b directly binds to the 3′UTR of Cbl-b.