P56 MiRNA-30 regulates the production of endogenous H2S by Cystathionine gamma-lyase (CSE) in primary cardiac myocytes

Abstract

Cystathionine gamma-lyase (CSE) is the major H2S-generating enzyme in myocardial cell. H2S has been reported to be cardioprotective during myocardial infarction (MI) injury. MiRNAs are identified as crucial gene regulators in response to myocardial infarction. However, there is little evidence indicating a functional role for miRNA in regulating the generation of H2S. The MI rat model was induced by ligating the left anterior descending coronary artery. Real-time PCR and western bolt results show that the expression of CSE mRNA and protein were increased in the infarct region and reduced in non-infarct and border zone regions 48 h after MI induction. The concentration of H2S in plasma was reduced significantly in MI model. Correspondingly, using real time PCR, we demonstrated that the miR-30 family, which includes 5 closely related miRNAs (miR-30a, miR-30b-5p, miR-30, miR-30d, miR-30e) were significantly down regulated in the infarct region and highly expressed in the non-infarct and border zone regions. To determine the potential roles of miR-30 family members in CSE gene expression, we transfected miRNA mimics into neonatal rat cardiomyocytes to upregulate the expression of miR-30 family numbers. Interestingly, overexpression of each number of the miR-30 family significantly repressed the protein expression of CSE tested by western blot and immunofluorescence, and reduced the H2S production in culture media. The mRNA expression of CSE was also inhibited by each number of the miR-30 family, respectively. In contrast, downregulation of all miR-30 family numbers by LNA modified miR-30 family inhibitor significantly increased the level of CSE mRNA and protein and the H2S concentration in culture media. We further demonstrated that miR-30 family members repressed CSE protein expression by directly targeting at CSE untranslational regions by luciferase activity assay. This result was consistent with computational prediction that CSE is a target of miR-30 family members. Take together, these results suggest that miR-30 family participate in regulating the production of endogenous H2S by targeting CSE.