MicroRNA ‐338‐3p as a therapeutic target in cardiac fibrosis through FGFR2 suppression

FIGURE 1. MiR‐338‐3p is downregulated in cardiac fibrosis. A. M‐mode cardiac ultrasound was performed to confirm left ventricular enlargement and heart failure in TAC mice. B. Gross and histological observations indicated that TAC mice showed prominent LV enlargement and myocardial fibrosis. C. Heat map of myocardial miRNAs with differential expression between the F and N groups (n = 3). D. MiR‐338‐3p amounts were decreased in heart tissue specimens from the F group (n = 3) compared with the N group (n = 3). E. MiR‐338‐3p amounts were reduced in left ventricular specimens from DCM patients (n = 7) in comparison with healthy controls (n = 8). F. MiR‐338‐3p was mainly expressed in C57BL/6 mice CFs. *, P < 0.05, **, P < 0.01 versus the corresponding control groups. DCM = dilated cardiomyopathy; F group = mouse model of left ventricular enlargement and heart failure established by transversal aortic constriction(TAC); N group = normal control; CF = cardiac fibroblast

FIGURE 2. MiR‐338‐3p overexpression suppresses CF activation, proliferation, and migration. A. α‐SMA, COL1A1, and COL3A1 mRNA amounts in AngII‐stimulated CFs after transfection with 50 nM miR‐338‐3p mimic/inhibitor and respective negative controls, normalized to ß‐Actin expression. B. Protein amounts of α‐SMA, COL1A, and COL3A1 in CFs after transfection with 50 nM miR‐338‐3p mimic/inhibitor and respective negative controls in the presence or absence of AngII stimulation (100 nM), normalized to GADPH expression. C. Immunofluorescent staining of α‐SMA (red) and F‐Actin (green) in AngII‐induced CFs after transfection with 50 nM miR‐338‐3p mimic or control. Scale bar = 25 μm. D. Proliferation analysis (CCK‐8 assay) of AngII‐treated CFs after transfection with 50 nM miR‐338‐3p mimic or negative control. E. Transwell migration assay of AngII‐treated CFs after transfection with 50 nM miR‐338‐3p mimic or negative control. Scale bar = 50 μm. *, p < 0.05, **, p < 0.01 versus respective controls. AngII = angiotensin II; α‐SMA = alpha smooth muscle Actin; CCK‐8 = Cell Counting Kit‐8; CFs = cardiac fibroblasts; COL1A1 = Collagen Type I Alpha 1 Chain; COL3A1 = Collagen Type 3 Alpha 1 Chain; FGFR2 =…

FIGURE 3. FGFR2 is a miR‐338‐3p target that blunts miR‐338‐3p‐mediated antifibrotic activity in CFs. A. Alignment of miR‐338‐3p and FGFR2 according to Targetscan (vertical bars), showing a high level of interaction and sequence conservation among mammalians. B and C. Luciferase activity assays demonstrated that the 3´‐UTR of FGFR2 was targeted by miR‐338‐3p. 293 T cells underwent transfection with FGFR2 3’‐UTR wild type reporter vector alongside miR‐338‐3p mimic and inhibitor, as well as respective controls (B). 293 T cells underwent transfection with FGFR2 3’‐UTR mutant reporter vector alongside 50 nM miR‐338‐3p mimic and inhibitor, as well as respective controls (C). D. Protein levels of FGFR2 were decreased significantly in CFs transfected with 50 nM miR‐338‐3p mimic. E. MiR‐338‐3p downregulated α‐SMA (red) and F‐Actin (green) in AngII‐induced CFs, which was reversed by FGFR2 overexpression. Scale bar = 25 μm. F. Protein amounts of α‐SMA and COL1A1 in AngII‐stimulated CFs after transfection with 50 nM miR‐338‐3p mimic and FGFR2‐overexpression plasmid, or respective negative controls. G. FGFR2 reversed the suppression of AngII‐treated CFs induced by 50 nM miR‐338‐3p (CCK8 assay). H.…

FIGURE 4. Serum miR‐338‐3p amounts at admission in individuals with or without cardiovascular adverse events during follow‐up. CV = cardiovascular. **, p < 0.01 versus respective controls