MiR-30e-5p Mitigates Hypoxia-induced Apoptosis In Human Stem Cell-derived Cardiomyocyocytes By Suppressing BIM

Coronary microembolization(CME)is considered a leading complication during percutaneous coronary intervention(PCI).CME can result in slow or no reflow.CME may also lead to cardiac contractile disfunction and myocardial arrhythmias,which are heavily related to the progression of infarct size and worsened patient condition at follow-up.Recently,microRNAs(miRNAs)have been implicated in the development of myocardial autophagy during CME.However,the underlying mechanism of these factors has not yet been fully elucidated.miRNAs are endogenous noncoding RNAs of 18–24 nucleotides that can negatively regulate gene expression at the posttranscriptional level via pair-bonding with the 3’-UTR of a target mRNA.Since one miRNA can target multiple genes,it has been found that miRNAs can participate in the regulation of diverse biochemical and physiological activities.Increasing evidence shows that miRNAs have diagnostic and therapeutic potential in cardiovascular diseases.Hypoxia can critically modify the miRNA profile in cardiomyocytes.Previous studies have also reported that autophagy is related to the occurrence and process of most cardiovascular diseases.Nevertheless,little evidencecurrently available has shown activated autophagy in cardiomyocytes during early myocardial hypoxia with treatment.In a previous study,we found several miRNAs that were markedly downregulated in rat cardiomyocytes after microsphere infusion.One of those miRNAs was miR-30e-5p,a miRNA that may participate in cardiomyocyte apoptosis and autophagy.The miR-30 family has recently been reported to participate in autophagy regulation.However,the role of miR-30e-5p in the development of cardiomyocyte apoptosis and autophagy after CME remains unclear.Cardiomyocytes are terminally differentiated cells and study of cardiomyocytes is hampered by the complexities in obtaining human heart tissue and the difficulties to propagate cardiac samples in culture.Consequently,most studies reported have used animal models to mimic cardiovascular disease.However,crucial biological and pathological differences exist between animal and human cardiac tissue,especially in cardiomyocytes.Recently,human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)have emerged as an appealing model for investigating cardiovascular diseases.Generation of hiPSC-CMs offers an innovative platform for direct assessment of cardiomyocyte function in vitro.Therefore,we utilized a novel hiPSC-CM model to mimic the development of hypoxia-induced injury during CME.Subsequently,the effect of miR-30e-5p on hiPSC-CM apoptosis and autophagy was investigated.Part 1 Generation of hiPSC-CMs Objective:To differentiate human induced pluripotent stem cells into cardiomyocytes and to identify properties and purity of cardiomyocytes.Methods:Human induced pluripotent stem cells were differentiated into cardiomyocytes in vitro,and the morphology of human induced pluripotent stem cells was observed.The hiPSC-CMs were identified by cellular immunofluorescence technique.Purity was detected by flow cytometry.Results:After passage of hiPSCs,the cells showed colony-like growth.When the confluency reached 90%,the hiPSCs were differentiated by the kit.Some cells contracted spontaneously from 7 days after induction,almost 90% of the cells beated vigorously after 10 days.The hiPSC-CMs were digested into individual cells and then inoculated and cultured for 40 days.To validate the efficiency of cardiomyocyte differentiation,immunostaining and flow cytometry were performed to examine the cardiomyocyte marker cTnT and α-actinin in hiPSC-CMs.It can be seen that the 40 days of hiPSC-CMs have well-defined and mature myofilament and Z-band structures.The results of flow cytometry showed the presence of cTnT in over 85% of the total cells.Part II miR-30e-5p downregulated in hiPSC-CMs after hypoxia-induced apoptosis.Objective:To explore the expression level of miR-30e-5p in hiPSC-CMs after hypoxia-induced apoptosis.Methods:According to the time of hypoxia treatment,the groups were 0h,6h,12 h,16h,20 h,and 24 h,and each group was independently repeated 3 times.The hypoxia model was constructed by placing hiPSC-CMs in a hypoxia incubator.The expression level of miR-30e-5p was investigated in hiPSC-CMs by RT-qPCR.The apoptosis level of hiPSC-CMs was detected by Caspase-3activity assay.Results:1.The expression level of miR-30e-5p in hiPSC-CMs was significantly downregulated in hypoxia at 12 h,16h,20 h and 24h(P < 0.05).2.Compared with the hypoxic 0h group,the caspase-3 activity of the hiPSC-CMs in the 24 h hypoxia group was remarkably upregulated(P < 0.05).Conclusion:The expression level of miR-30e-5p was downregulated in hiPSC-CMs after hypoxia-induced apoptosis.Part III miR-30e-5p targeted BIM to inhibit hypoxia-induced apoptosis in hiPSC-CMs Objective:To investigate the role of miR-30e-5p in hiPSC-CMs after hypoxia-induced apoptosis,and to explore the mechanism of miR-30e-5p targeting BIM in hiPSC-CMs.Methods:40 days of hiPSC-CMs were divided into normal group(N),hypoxia group(H),negative control group(NC+H)and miR-30e-5p overexpression group(miR+H).The N group was cultured in a normal incubator.The H group was placed in a hypoxia incubator for 24 h.The NC+H and miR+H groups were stably transfected with a recombinant lentivirus loaded with miR-NC and miR-30e-5p,respectively.The expression levels of miR-30e-5p and BIM mRNA in hiPSC-CMs were explored by RT-qPCR.The apoptosis level of hiPSC-CMs was detected by Caspase-3 activity assay and flow cytometry.Calcium fluorescence was used to examine the calcium transient level of hiPSC-CMs.Western blot was performed to detect the expression levels of apoptosis-related proteins Bax,Bcl-2,Caspase-3 and target protein BIM.The target gene of miR-30e-5p was predicted using a bioinformatics database and verified by a dual luciferase reporter gene assay.Results:1.Compared with N group,the expression level of miR-30e-5p in group H was downregulated.Compared with NC+H group,the expression level of miR-30e-5p in miR+H group was significantly upregulated.2.Compared with the N group,the activity level of Caspase-3 in the Hgroup was increased.Compared with the NC+H group,the activity level of Caspase-3 in the miR+H group decreased.3.Compared with N group,the ratios of apoptosis in hPS-CMs in group H increased.Compared with NC+H group,the ratios of apoptosis in hiPSC-CMs in miR+H group decreased.4.Compared with the N group,the amplitude and decay time of the calcium transients in the H group hiPSC-CMs were prolonged.Compared with the NC+H group,the amplitude and decay time of the calcium transients of the hiPSC-CMs in the miR+H group were shortened.5.Compared with group N,the expression levels of apoptosis-related proteins Bax/Bcl-2 and Caspase-3 were upregulated in group H.Compared with NC+H group,the expression level of apoptosis-related proteins Bax/Bcl-2 and Caspase-3 in miR+H group were downregulated.6.Compared with the N group,the expression level of BIM protein in group H was upregulated;compared with the NC+H group,the expression level of protein BIM in miR+H group was downregulated.7.Bioinformatics database analysis showed that BIM is one of the potential target genes of miR-30e-5p.Dual luciferase reporter gene assays confirmed that miR-30e-5p can directly bind to the 3 UTR sequence of BIM,resulting in the luciferase activity was reduced,whereas the corresponding BIM 3 UTR mutation binding site had no effect on luciferase activity.Conclusion:1.miR-30e-5p regulated hypoxia-induced apoptosis in hiPSC-CMs.2.BIM is one of the target genes of miR-30e-5p3.miR-30e-5p targeted BIM to inhibit hypoxia-induced apoptosis in hiPSC-CMsPart IV BIM inhibited hypoxia-induced apoptosis in hiPSC-CMs by activating autophagy Objective:To elucidate the potential mechanism of BIM in the regulation of hypoxia-induced apoptosis in hiPSC-CMs.Methods:The expression levels of autophagy-related proteins Beclin-1 and p62 were examined in NC+H and miR+H groups.The 40-day hiPSC-CMs were divided into Control shRNA+H group(shNC+H)and shRNA BIM+H group(shBIM+H),shNC+H group and shBIM+H group were stably transfected with recombinant shRNA containing Control shRNA and BIM shRNA for 24 h after hypoxia treatment.Apoptosis levels of hiPSC-CMs were analyzed by Caspase-3 viability assays and flow cytometry.Calcium fluorescence was performed to detect the calcium transient level of hiPSC-CMs.Western blot was used to measure the expression levels of target protein BIM,apoptosis protein Caspase-3 and autophagy related proteins Beclin-1 and p62.Results:The expression levels of the autophagy-related proteins Beclin-1 and p62 were increased in the miR+H group compared with the NC+H group.After inhibiting the expression level of BIM,comparing with shNC+H group,caspase-3 activity,apoptosis ratios and apoptosis protein Caspase-3 decreased in shBIM+H group,while the expression level of autophagy-related proteins Beclin-1 and p62 increased.Conclusion:BIM inhibited hypoxia-induced apoptosis in hiPSC-CMs by activatingautophagy.