Figure 5.

circHNRNPH1 Mediated Primary Cardiac Fibroblast Apoptosis by Sponging miR216-5p

(A) FISH experiment showing that circHNRNPH1 colocalized with miR216-5p in the cytoplasm of primary cardiac fibroblasts. Red, circHNRNPH1; green, miR216-5p; blue, 4′,6-diamidino-2-phenylindole (DAPI). The images are representative of three independent experiments. Magnification 400×. Scale bars, 50 μm. (B) Schematic graphics of the pmirGLO plasmids with circHNRNPH1-WT and circHNRNPH1-Mut for the dual-luciferase reporter assay. (C) Dual-luciferase reporter assay. The luciferase activity was analyzed by quantitative PCR. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). (D) Diagram of the circHNRNPH1 sponge with biotin-coupled miR-216-5p WT and its Mut (top). (Bottom) The WT (bio-216-wt) and Mut (bio-216-mut) biotin-coupled miR-216-5p sequences are shown. (E) The WT (bio-216-wt) or Mut (bio-216-mut) biotin-coupled miR-216-5p was transfected into primary cardiac fibroblasts. After streptavidin capture, circHNRNPH1 and GAPDH mRNA were quantified by quantitative PCR, and the relative pellet/input ratio was plotted. Input: 10% samples were loaded; pellet: 100% samples were loaded. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). (F and G) RIP assay. AGO2 RNA immunoprecipitation (IP) in primary cardiac fibroblasts transfected with circHNRNPH1 or its Mut (F) or with miR216-5p or its Mut (G). The level of circHNRNPH1 or miR216-5p was quantified by quantitative PCR. RIP represents an RNA-binding protein IP assay; input: 10% samples were loaded. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). (H) Quantitative PCR examination of the expression of SMAD7 regulated by circHNRNPH1. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). (I and J) Western blot (I) and densitometric analysis (J) of SMAD7 expression regulated by circHNRNPH1. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). (K and L) Western blot (K) and densitometric analysis (L) of SMAD7 expression regulated by circHNRNPH1 via miR216-5p. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). (M and N) Western blot (M) and densitometric analysis (N) of apoptotic proteins in primary cardiac fibroblasts with circHNRNPH1 overexpression or knockdown. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). (O and P) TUNEL assay (O) and quantitative analysis (P) of primary cardiac fibroblasts with circHNRNPH1 overexpression or knockdown. si-NC, si-circHNRNPH1-NC; si-circ, si-circHNRNPH1; circ-NC, circHNRNPH1-NC; circ, circHNRNPH1; DAPI, 4′,6-diamidino-2-phenylindole. Data are presented as the mean ± SD (n = 3 per group, ∗p < 0.05). The images are representative of three independent experiments. Green, TUNEL-positive cells; blue, DAPI. Magnification 200×. Scale bars, 50 μm. (Q) Primary cardiac fibroblast apoptosis assay by flow cytometry with overexpression or knockdown of circHNRNPH1. si-NC, si-circHNRNPH1-NC; si-circ, si-circHNRNPH1; circ-NC, circHNRNPH1-NC; circ, circHNRNPH1.