miR-21 enhances the protective effect of loperamide on rat cardiomyocytes against hypoxia/reoxygenation, reactive oxygen species production and apoptosis via regulating Akap8 and Bard1 expression

Hong Shen, Zhifeng Yao, Weipeng Zhao, Yaping Zhang, Chenling Yao, Chaoyang Tong
Experimental and Therapeutic Medicine 2019, 17 (2): 1312-1320
Effective therapies to reduce ischemia/reperfusion and hypoxia/reoxygenation injury are currently lacking. Furthermore, the effects of loperamide and microRNA (miR)-21 on hypoxia/reoxygenation injury of cardiomyocytes have remained to be elucidated. Therefore, the present study aimed to investigate the effect of loperamide and miR-21 on cardiomyocytes during hypoxia/reoxygenation injury, and to explore the underlying molecular mechanisms. H9c2 rat cardiomyocytes were pre-treated with loperamide prior to hypoxia/reoxygenation. The viability of H9c2 cells was measured with a cell counting kit 8 and apoptosis was detected with an Annexin V-phycoerythrin/7-aminoactinomycin D apoptosis kit. Furthermore, reactive oxygen species were detected with a specific kit. Genes regulated by miR-21 were screened with an mRNA chip and confirmed using reverse-transcription quantitative polymerase chain reaction analysis. The direct targeting relationship of miR-21 with certain mRNAs was then confirmed using a Dual-Luciferase Reporter Assay system. The results indicated that the apoptotic rate and reactive oxygen species levels in rat cardiomyocytes were markedly increased after hypoxia/reoxygenation treatment. Pre-treatment with loperamide significantly protected H9c2 cells against apoptosis and reactive oxygen species production after hypoxia/reoxygenation. The protection was markedly decreased by miR-21 inhibitor and enhanced by miR-21 mimics. Screening for genes associated with cardiomyocyte apoptosis revealed that the relative expression of A-kinase anchoring protein 8 (Akap8) and BRCA1 associated RING domain 1 (Bard1) was consistent with the experimental results on apoptosis and reactive oxygen species. Compared with the group treated by hypoxia/reoxygenation alone, pre-treatment with loperamide markedly decreased the expression of BRCA1-interacting protein C-terminal helicase 1, Akap8 and Bard1 after hypoxia/reoxygenation. The decrease in the expression of Akap8 and Bard1 was markedly attenuated by miR-21 inhibitor and enhanced by miR-21 mimics. miR-21 mimics directly targeted the 3'-untranslated region (UTR) of Akap8 and Bard1 mRNA to thereby decrease their expression. In conclusion, the protection of rat cardiomyocytes against hypoxia/reoxygenation-induced apoptosis and reactive oxygen species production by loperamide was markedly enhanced by miR-21. miR-21 directly targets the 3'-UTR of Akap8 and Bard1 mRNA and enhances the inhibitory effects of loperamide on Akap8 and Bard1 expression in rat cardiomyocytes after hypoxia/reoxygenation.

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