MiR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression

Figures

• 

  Figure 1. MiR-21 overexpression and HIPK3 down-regulation increase cell viability and decrease LDH activity (A) The cell viability relative to CTL: miR-21 overexpression or HIPK3 down-regulation increased cell viability while miR-21 suppression decreased cell viability (n=3). (B) LDH activity: miR-21 overexpression or HIPK3 down-regulation decreased LDH activity while miR-21 suppression increased LDH activity (n=3 or 4). (C) HIPK3 mRNA expression relative to CTL (n=3). Data were presented as mean ± SD; ##P<0.01 vs. CTL; **P<0.01 vs. H/R+ 21mimic NC; +P<0.05 vs. H/R+ 21inhibitor NC; ++P<0.01 vs. H/R+ 21inhibitor NC; ΔΔP<0.01 vs. H/R+ si-NC.

• 

  Figure 2. MiR-21 overexpression and HIPK3 down-regulation suppress apoptosis (A) TUNEL staining showed miR-21 overexpression or HIPK3 down-regulation significantly reduced TUNEL positive cells, and miR-21 suppression increased TUNEL positive cells (×400 magnification). (B) Representative images of flow cytometry: miR-21 overexpression or HIPK3 down-regulation inhibited apoptosis, and miR-21 suppression promoted apoptosis. (C) Quantitative analysis of TUNEL positive cells (n=3). (D) Rates of apoptosis cells detected by flow cytometry (n=3). Data are presented as mean ± SD; ##P<0.01 vs. CTL; **P<0.01 vs. H/R+21mimic NC; ++P <0.01 vs. H/R+ 21inhibitor NC; ΔΔP<0.01 vs. H/R+si-NC.

• 

  Figure 3. MiR-21 affects apoptosis-related proteins (A) Representative images of Western blot analysis. (B) and (C) Statistical analysis of protein levels: miR-21 overexpression decreased BAX expression and increased BCL2 expression, and miR-21 suppression increased BAX expression and decreased BCL2 expression. The gray values of bands were normalized to that of beta Actin (n=3). Data are presented as mean ± SD; ##P<0.01 vs. CTL; *P<0.05 vs. H/R+ 21mimic NC; **P<0.01 vs. H/R+ 21mimic NC; ++P<0.01 vs. H/R+ 21inhibitor NC.

• 

  Figure 4. MiR-21 interacts with HIPK3 (A) Representative immunofluorescence images of HIPK3 expression levels (×400 magnification). (B) Representative images of Western blot analysis. (C) Statistical analysis of HIPK3 levels: miR-21 overexpression inhibited HIPK3 expression, and miR-21 suppression up-regulated HIPK3 expression. The gray values of bands were normalized to that of beta Actin (n=3). (D) MiR-21 expression relative to CTL: transfecting with si-HIPK3 increased miR-21 expression, and transfecting with miR-21 mimic increased miR-21 expression while transfecting with miR-21 inhibitor decreased miR-21 expression (n=3–5). Data are presented as mean ± SD; #P<0.05 vs. CTL; ##P<0.01 vs. CTL; *P<0.05 vs. H/R+ 21mimic NC; **P<0.01 vs. H/R+21mimic NC; +P<0.05 vs. H/R+21inhibitor NC; ++P<0.01 vs. H/R+ 21inhibitor NC; ΔΔP<0.01 vs. H/R+ si-NC.

• 

  Figure 5. MiR‐21‐5p directly binds to 3′‐UTR of HIPK3 (A) The binding site of miR‐21 with HIPK3 3′‐UTR was predicted in the TargetScan 7.2 website. (B) Luciferase activity of HIPK3‐WT‐3′‐UTR or HIPK3‐MUT‐3′‐UTR after cotransfecting miR-21 mimic was detected by dual luciferase reporter assay. Luciferase activity of H9C2 cells cotransfected with miR-21 mimic and psiCHECK™-2 vector containing HIPK3-WT-3′-UTR significantly decreased (n=3). Data are presented as mean ± SD; **P<0.01; NS, no significance.

• 

  Figure 6. HIPK3 down-regulation affects the expression of proteins relative to Fas-mediated apoptosis (A) Representative images of Western blot analysis. (B–F) Statistical analysis of protein levels: HIPK3 down-regulation decreased the phosphorylation of FADD and the expression of BAX and C-Caspase-3, and increased the expression of BCL2 (n=3). In panels B,D,E, the gray values of bands were normalized to that of beta Actin. C-Caspase-3, cleaved caspase-3; p-FADD, phosphorylated FADD. Data are presented as mean ± SD; #P<0.05 vs. CTL; ##P<0.01 vs. CTL; ΔP<0.05 vs. H/R+ si-NC; ΔΔP<0.01 vs. H/R+ si-NC.