Figure 3.

miR-132 binds to the 3′UTR of TGF-β2 to inhibit its expression. (A) Schematic of the miR-132 binding site in the TGF-β2 3′UTR at position 746-753 and its sequence homology to miR-132. The mutant version of the TGF-β2 3′UTR is shown. (B) The wt and mt TGF-β2 3′UTRs were cloned into a pLightSwitch Renilla plasmid and transfected into AsPC-1, PANC-1 and ASAN-PaCa cells in the presence or absence of 50 nM miR-132 mimics. Negative mimics served as a control. Co-transfection with Firefly luciferase (0.25 ng/µl) served as a normalization control. At 48 h after transfection, the expression of Renilla and Firefly luciferases was detected using a FLUOstar Omega microplate reader. Renilla luciferase activities were normalized to Firefly luciferase activities. (C) PANC-1 cells were transfected with miR-132 or non-coding miRNA (NC), or mock-treated without miRNA (CO). Proteins were harvested 48 h later and analyzed by western blotting. β-actin served as the normalization control. (D) Representative paraffin-embedded PDA tissue sections from patients with documented pre-operative intake of inhaled (n=8) or oral (n=6) GCs (+GC, n=14) and from those without GC intake (−GC, n=20) were evaluated by IHC to detect the expression of TGF-β2. A semi-quantitative scoring system was used to evaluate expression levels based on visual determination of the percentage of positive cells. The sections were analyzed at ×400 magnification, and representative images are shown. (E) In situ hybridization of miR-132 expression in patient tissues. The arrows indicate positive cells. Representative results for part B are shown as the means ± SD. ^*P<0.05. UTR, untranslated region; wt, wild-type; mt, mutant/mutated; miR, microRNA; CO, control/untreated; GC, glucocorticoids.