MicroRNA‐129‐1‐3p protects cardiomyocytes from pirarubicin‐induced apoptosis by down‐regulating the GRIN2D‐mediated Ca²⁺ signalling pathway

Figure 1. THP induces cardiomyocyte injury and down‐regulates miR‐129‐1‐3p. (A, B) H9C2 (A) and HL‐1 (B) cells were incubated with THP at indicated concentrations for 24 h. Cell viability was evaluated using the CCK‐8 assay. (C) H9C2 and HL‐1 cells were incubated with 5 μmol/L THP for 24 h. Representative microscopic images are shown. (D, E) H9C2 (D) and HL‐1 (E) cells were incubated with 5 μmol/L THP for 24 h. MiR‐129‐1‐3p transcript levels were determined using qRT‐PCR. n = 3, *P < .05 and **P < .01 vs Control

Figure 2. MiR‐129‐1‐3p alleviates THP‐induced ROS production in cardiomyocytes. (A, B) H9C2 (A) and HL‐1 (B) cells were transfected with the miR‐129‐1‐3p mimics or miR‐129‐1‐3p inhibitor for 8 h. Representative fluorescence images (×40 magnification, left) and quantified transfection efficiency (right, n = 3) are shown. (C) H9C2 and HL‐1 cells were transfected as indicated and treated with 5 μmol/L THP or vehicle alone for 24 h. Un‐transfected cells were included for comparison. Intracellular ROS levels were evaluated using the DCFH‐DA staining assay. Representative fluorescence images are shown (×100 magnification)

Figure 3. MiR‐129‐1‐3p protects cardiomyocytes from THP‐induced apoptosis. H9C2 and HL‐1 cells were transfected as indicated and treated with 5 μmol/L THP or vehicle alone for 24 h. Un‐transfected cells were included for comparison. (A, F) Cell apoptosis was evaluated using the TUNEL assay. Representative fluorescence images (×100 magnification, left) and quantified apoptosis rates (right) are shown. (B, G) Cell apoptosis was assessed using flow cytometry. Representative histograms (upper panel) and quantified apoptosis rates (lower panel) are shown. (C‐E) mRNA levels of Caspase‐3, Bax and Bcl‐2 in H9C2 cells were determined using qRT‐PCR. (H‐J) mRNA levels of Caspase‐3, Bax and Bcl‐2 in HL‐1cells were determined using qRT‐PCR. n = 3; **P < .01 vs Control; # P < .05 and ## P < .01 vs THP

Figure 4. MiR‐129‐1‐3p is predicted to regulate the Ca2+ pathway by directly targeting GRIN2D. A, GO enrichment analysis of miR‐129‐1‐3p and its target genes. B, Diagram of tentative binding between miR‐129‐1‐3p and the 3′‐UTR of rat or mouse GRIN2D mRNA. C, KEGG pathway enrichment analysis of miR‐129‐1‐3p. D, The top 20 pathways dysregulated following THP‐induced myocardial injury

Figure 5. MiR‐129‐1‐3p directly targets GRIN2D. A, Diagram of the GRIN2D 3′‐UTRs containing the wild‐type or mutant miR‐129‐1‐3p‐binding site (left/upper), a diagram of the luciferase reporter vector (right) and the luciferase reporter assay data (left/lower) are shown. (B–E) H9C2 (B, D) and HL‐1 (C, E) cells were transfected as indicated and treated with 5 μmol/L THP or vehicle alone for 24 h. Un‐transfected cells were included for comparison. The levels of GRIN2D mRNA (B, C) and miR‐129‐1‐3p transcript (D, E) were determined by qRT‐PCR. n = 3; *P < .05 and **P < .01 vs Control; # P < .05 and ## P < .01 vs THP

Figure 6. MiR‐129‐1‐3p inhibits THP‐induced calcium imbalance in cardiomyocytes. H9C2 (A, C) and HL‐1 (B, D) cells were transfected as indicated and treated with 5 μmol/L THP or vehicle alone for 24 h. Un‐transfected cells were included for comparison. (A, B) Intracellular Ca2+ levels were assessed using fluorescence imaging with Fluo‐3 AM staining. Representative fluorescence images (×100 magnification, upper panel) and percentages of Fluo‐3 AM‐positive cells (lower panel) are shown. (C, D) Protein expression of GRIN2D, CALM1, CaMKⅡδ, SERCA2a, RyR2, RyR2‐pS2814, NCX1, Bax and Bcl‐2 were determined using Western blot analysis. n = 3; *P < .05 and **P < .01 vs Control; # P < .05 and ## P < .01 vs THP