MicroRNA-214-5p protects against myocardial ischemia reperfusion injury through targeting the FAS ligand

Figures

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  Figure 1. The expression of miR-214-5p was decreased in H9c2 cells under H/R conditions. The expression of miR-214-5p in H9c2 cells under H/R treatment was detected by qRT-PCR Data are presented as mean ± SD. ***P < 0.001.

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  Figure 2. miR-214-5p attenuated H/R treatment induced cardiomyocyte injury. H9c2 cell lines were transfected with miR-214-5p mimic or inhibitor for 24 h followed by H/R treatment. A – The effect of miR-214-5p mimic or inhibitor on the expression level of miR-214-5p was detected by qRT-PCR in H9c2 cells under H/R conditions. B – The effect of up- or down-regulation of miR-214-5p expression on the LDH activity was measured by LDH assay in cardiac cells upon H/R treatment conditions. C – The role of overexpression or downregulation of miR-214-5p on the MDA activity was determined by MDA detection assay Data are presented as mean ± SD. ***P < 0.001.

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  Figure 3. miR-214-5p reduced H/R-induced ROS production in cardiomyocytes. The function of elevated or reduced expression of the miR-214-5p by its mimic or inhibitor at the ROS generation level was evaluated by ROS detection assay after H9c2 cells’ insult due to H/R injury Data are presented as mean ± SD. ***P < 0.001.

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  Figure 4. miR-214-5p suppressed apoptosis of cardiac cells subject to H/R impairment. A – The effect of overexpression or silencing of miR-214-5p in H9c2 cells after H/R treatment on apoptosis rate of cardiac cells was tested by cell apoptosis analysis using a flow cytometer. B – The effect of enhancing or interference of miR-214-5p expression levels on the anti-apoptotic proteins Bcl-2 or pro-apoptotic proteins including Bax, cleaved-caspase 3 and cleaved-caspase 9 was determined by western blotting in H9c2 cells after H/R injury Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 5. FASLG was targeted by miR-214-5p. A – The 3′-UTR of FASLG mRNA containing the predicted target sites for miR-214-5p (FASLG-3′-UTR-wt) or the mutated miR-214-5p binding sites in the FASLG gene (FASLG-3′-UTRmut) are shown. B – Luciferase activities of H9c2 cells co-transfected with miR-214-5p mimics or NC mimics and luciferase reporters containing FASLG 3′-UTR-wt or -mut transcript were checked by dual-luciferase reporter assays. C – The effect of up- or down-regulated expression of miR-214-5p on the protein level of FASLG was measured by western blot in H/R-treated H9c2 cells Data are presented as mean ± SD. ***P < 0.001.

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  Figure 6. The protective role of miR-214-5p on the H/R injured cardiac cells was blocked by overexpression of FASLG. The H9c2 cells were co-transfected with miR-214-5p mimic along with FASLG pcDNA followed by H/R treatment. A – The influence of overexpression of miR-214-5p and up-regulation of FASLG on the LDH and MDA activities was tested by LDH and MDA assay kit in H9c2 cells under H/R treatment. B – The effects of enhanced miR-214-5p level and elevated expression of FASLG on ROS production were evaluated by ROS detection assay in H9c2 cells upon H/R exposure. C – The effects of up-regulated miR-214-5p level and enhanced FASLG expression on the apoptosis rate were determined by cell apoptosis analysis assay in H9c2 cells by H/R treatment. D – The effect of advanced expression level of miR-214-5p when co-transfected with FASLG overexpression construct in H9c2 cells on the protein levels including FASLG, Bcl-2, Bax, cleaved-caspase 9 and cleaved-caspase3 was measured by western blotting upon H/R exposure Data are presented as mean ± SD. ***P < 0.001.

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  Figure 7. miR-214-5p reduced cardiac cell damage induced by I/R injury in mice. The animals were administered with Ad-miR-214-5p in an intracoronary delivery manner 5 days before the I/R operation. A – The effect of adenovirus on the expression level of miR-214-5p was evaluated by qRT-PCR in I/R-treated mice. B, C – The influence of overexpression of miR-214-5p on the CK and LDH activity was examined by CK and LDH assay kit in mice exposed to I/R treatment. D – The influence of Ad-miR-214 on the focal ischemia of heart slices and quantitative analysis of heart infarct size were assessed by TTC staining in I/R-treated animals. E – Representative images of cardiac tissue slices were acquired by HE staining in mice injected with Ad-miR-214-5p following I/R treatment. F – The effect of enhanced miR-214-5p expression on the pro- or anti-apoptotic protein levels was evaluated by western blotting in mice upon I/R exposure. N = 6 per group Data are expressed as mean ± SD; **p < 0.01, ***p < 0.001.