RESUMO
Aim To screen the differentially expressed microRNAs ( miRNAs ) induced by hypoxia precondi-tioning ( HPC ) in adult rat cardiomyocytes, and pre-dict miRNAs-regulated target genes and their func-tions. Methods Cardiomyocytes were isolated from a-dult rat ventricular myocardium and cultured ( in vitro) . The cells were divided into 2 groups: control group ( CON ) and hypoxia preconditioning group ( HPC) . The cardiomyocytes in HPC group were sub-jected to 10 min hypoxia followed by 30 min reoxygen-ation, while the cells in CON group were cultured un-der normal condition. After that, total RNA was ex-tracted and then subjected to miRNA microarray to screen differentially expressed miRNAs. The microar-ray results were further validated by quantitative RT-PCR ( qRT-PCR ) . Bioinformatics analysis was per-formed to predict the miRNAs-regulated target genes and analyze the enriched gene ontology ( GO) and sig-naling pathway ( Pathway) . Results HPC caused sig-nificant changes in miRNAs expression in cardiomyo-cytes as compared to CON group. A total of 12 miR-NAs were up-regulated and 14 miRNAs were down-reg-ulated ( P 500 were selected for further bioinformatics analysis. The expression of miR-133b-5p, miR-664-1-5p and miR-6216 detected by qRT-PCR exhibited the similar patterns of up or down regulation to those shown in mi-croarray results. Bioinformatics analysis revealed that miRNAs-regulated target genes were significantly en-riched in 27 GOs and 6 signal pathways. Conclusion The expression profile of miRNAs in rat cardiomyo-cytes is significantly affected by HPC. These differenti-ally expressed miRNAs might participate in HPC-in-duced cardioprotection by regulating their target genes in rat cardiomyocytes.
RESUMO
Objective To evaluate the effect of morphine preconditioning on the expression of miR-133b-Sp and Fas in rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R).Methods Cardiomyocytes were isolated from healthy adult male Sprague-Dawley rats by using Langendorff perfusion.The cells were seeded into 24-well plates or 60 mm diameter dishes and randomly divided into 3 groups (n =24 each) using a random number table:control group (group C),group H/R,and morphine preconditioning group (group MPC).The cells in group C were cultured in normal culture atmosphere.In H/R and MPC groups,the cells were exposed to 95％ N2-5％ CO2 for 90 min followed by 120 min reoxygenation.In group MPC,the cells were cultured for 10 min in serum-free DMEM liquid culture medium containing morphine 1 μmol/L,and then were cultured for 30 min in morphine-free DMEM liquid culture medium before hypoxia.At 120 min of reoxygenation,the cells in 24-well plates were selected to detect the cell viability (by MTT),lactate dehydrogenase (LDH) activity in the culture medium,and cell apoptosis (by Hoechst 33234 staining).Apoptosis rate was calculated.Total RNA and protein were extracted from the cells in 60 mm dishes to detect the expression of miR-133b-5p and Fas mRNA (by quantitative real-time PCR) and Fas protein (by Western blot).Results Compared with C group,the cell viability was significantly decreased,LDH activity and apoptosis rate were increased,the expression of miR-133b-Sp was down-regulated,and the expression of Fas mRNA and protein was up-regulated in H/R group.Compared with H/R group,the cell viability was significantly increased,LDH activity and apoptosis rate were decreased,the expression of miR-133b-5p was up-regulatcd,and the expression of Fas mRNA and protein was down-regulated in MPC group.Conclusion The mechanism by which morphine preconditioning reduces H/R injury to rat cardiomyocytesis related to up-regulation of the expression of miR-133b-Sp and down-regulation of the expression of Fas.