MiR ‐129‐5p alleviates myocardial injury by targeting suppressor of cytokine signaling 2 after ischemia/reperfusion

FIGURE 1. The expression of miR‐129‐5p was downregulated in myocardial tissues of mouse I/R model. A, The serum LDH activity and MDA content were evaluated using commercial kit following the manufactures' protocol. B, The miR‐129‐5p level in sham and I/R groups was measured by quantitative real‐time PCR. C, The infract size of mouse myocardial tissues was measured by TTC staining (infract area/area at risk). I/R, ischemia/reperfusion; LDH, lactate dehydrogenase; MDA, malondialdehyde; TTC, triphenyl tetrazolium chloride. **P < .01

FIGURE 2. MiR‐129‐5p overexpression alleviated I/R induced myocardial injury. Mice were intravenously injected with miR‐129‐5p agomir and NC agomir every 3 days for 2 weeks before surgery.A, The miR‐129‐5p expression after agomir injection was determined by quantitative real‐time PCR.B, The serum LDH activity and MDA content after agomir injection were assessed using commercial kit. C, The myocardial injury was evaluated by TCC staining. I/R, Ischemia/reperfusion; LDH, Lactate dehydrogenase; MDA, Malondialdehyde; NC, nonspecific control; TTC, triphenyl tetrazolium chloride. **P <.01

FIGURE 3. MiR‐129‐5p overexpression ameliorated H9C2 cell injury induced by H/R. H9C2 cells were cultured under hypoxic condition for 6 hours and transferred to normal oxygen condition to induce in vitro H/R model, followed by miR‐129‐5p mimics/NC mimics transfection. A, The relative miR‐129‐5p level in H9C2 cells after H/R was measured by quantitative real‐time PCR. B, The H9C2 cell viability after H/R was assessed using cell‐counting kit‐8 assay. C, The LDH activity and MDA content in H/R induced H9C2 cells were determined using commercial kit. D, H9C2 cell apoptosis after H/R was detected using flow cytometry. H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; MDA, malondialdehyde; NC, nonspecific control. **P < .01

FIGURE 4. SOCS2 was a downstream target of miR‐129‐5p. A, The binding site of miR‐129‐5p on SOCS2 was shown. B, The luciferase reporter assay was performed to validate the interaction between miR‐129‐5p and SOCS2. C, The mRNA level of SOCS2 in H/R induced H9C2 cells after miR‐129‐5p mimics transfection was measured by quantitative real‐time PCR. D, The protein level of SOCS2 in H/R induced H9C2 cells after miR‐129‐5p mimics transfection was determined by western blot assay. H/R, hypoxia/reoxygenation; SOCS2, suppressor of cytokine signaling 2. **P < .01

FIGURE 5. MiR‐129‐5p overexpression alleviated H/R induced H9C2 cell injury by targeting SOCS2. H/R induced H9C2 cells were cotransfected with pcDNA3.1‐SOCS2 and miR‐129‐5p mimics. A, Cell viability was assessed using cell counting kit‐8 assay. B, Commercial kit was used to determine the LDH activity and MDA content in H/R induced H9C2 cells. C, The protein levels of cleaved PARP and cleaved caspase‐3 in non‐H/R induced H9C2 cells were detected using western blot. D, The protein levels of SOCS2, cleaved PARP and cleaved caspase‐3 in H/R induced H9C2 cells were determined by western blot. H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; MDA, malondialdehyde; PARP, Poly‐(ADP‐ribose) polymerase; SOCS2, suppressor of cytokine signaling 2. **P < .01