Figure 2.

MiR-26a targets TRPC3 and TRPC6 and is reduced in AP

(A) Schematic of human TRPC3 and TRPC6 and mouse Trpc3 and Trpc6 3′ UTRs. Locations of the predicted miR-26a binding sites are indicated. (B) Relative luciferase activity in HEK293T cells transfected with reporter constructs containing the WT or mutated (Mut) 3′ UTR of target genes and co-transfected with either miR-26a mimic (miR-26a) or negative control (NC). (C) Protein levels of Trpc3 and Trpc6 in PACs of 26a DKO, Hprt Tg, and control mice. (D and E) Levels of miR-26a in the pancreata with CER-AP (7 cerulein injections) at 12 h (D) and TLCS-AP (5 mM TLCS) at 24 h (E). (F and G) Levels of miR-26a in mouse PACs stimulated with CCK (100 nM) for 1 h (F) and TLCS (500 μM) for 30 min (G). (H and I) Expression of miR-26a (H) and representative immunohistochemical staining (scale bar, 50 μm; magnification ×200) of TRPC3 and TRPC6 (I) in AP and normal pancreata (Ctrl). Black outlines: higher magnification of the indicated pancreata area in the low magnification images by black squares (scale bars, 20 μm; magnification ×400). TRPC3 and TRPC6-positive area were quantified per five high-powered fields with ImageJ software. (J) Expression of miR-26a in AP pancreata of patients with sterile or infected necrosis. (K and L) Serum levels of miR-26a in CER-AP (K) and TLCS-AP (L) from (D and E), respectively. (M) Level of plasma miR-26a in healthy volunteers (left) and AP patients on admission (middle) and at 72 h of hospitalization (right). (N) Heapmap depicting Spearman correlation between admission miR-26a and routine clinical biomarkers for severity assessment. Data are from 6–13 mice per group in (D)–(G), n = 10 (AP) and n = 7 (Ctrl) in (H) and (I), n = 6 (sterile necrosis) and n = 4 (infected necrosis) in (J), n = 27 (healthy volunteers) and n = 85 (AP patients) in (M), and shown as mean ± SEM.