Fig. 4.

Cbl-b is a direct target of miR-29b-2-5p and involved in miR-29b-2-5p-induced tumor suppression. a Target site of miR-29b on 3UTRs of Cbl-b mRNA. The wild-type and mutated constructs were shown with the green and red seed region in bold. b Luciferase activity of pMirTarget-Cbl-b-wt or pMirTarget-Cbl-b-mut in Capan-2 and SW1990 cells after transfection with miR-29b-2-5por control. The error line represents the mean ± SD, *P < 0.05. c miR-29b-2-5p inhibited the expression of Cbl-b at the post-transcriptional level. SW1990 and Capan-2 were transfected with miR-29b-2-5p mimic in different concentrations. Western blot indicated miR-29b-2-5p down-regulated the expression of Cbl-b protein. RT-PCR suggested overexpression of miR-29b-2-5p did not significantly affect the level of Cbl-b mRNA; n = 3. d PDAC cell lines SW1990 and Capan-2 were transfected with p3xFLAG-CMV9-cbl-b (OE Cbl-b) or p3Xflag-CMV9(NC). Overexpression effect of Cbl-b was examined by Western blot; n = 3. e Cells were collected at 48, 24, 72, and 96 h after transfection using Trypan blue staining method. Take the 24 h/24 h, 48 h/24 h, 72 h/24 h, 96 h/24 h ratio respectively. The results suggested Cbl-b significantly promote the proliferation of PDAC cells (mean ± SD, results of three independent experiments, *P < 0.05). f SW1990 was co-transfected with a control nonspecific mimic (NC), miR-29b-2-5p, NC + p3xFLAG-CMV9-cbl-b and p3xFLAG-CMV9-cbl-b + miR-29b-2-5p. The results showed that miR-29b-2-5p could effectively reverse the effect of Cbl-b on the proliferation of PDAC cells