MicroRNAs (miRs) are a class of important regulators, which are involved in the regulation of apoptosis. Oxidative stress-induced apoptosis is the predominant factor accounting for cardiac ischemia-reperfusion injury. miR-153 has been previously shown to have an antitumor effect in cancer. However, whether miR-153 is involved in oxidative stress-induced apoptosis in the heart remains to be elucidated. To this end, the present study used reverse transcription-quantitative polymerase chain reaction to detect miR-153 levels upon oxidative stress, and evaluated apoptosis, autophagy and expression of critical genes by western blotting. A luciferase assay was also used to confirm the potential target gene. In the present study, it was found that the expression of miR-153 was significantly increased upon H2O2 stimulation, and the inhibition of endogenous miR-153 decreased apoptosis. To further identify the mechanism underlying the pro-apoptotic effect of miR-153, the present study analyzed the 3′untranslated region of myeloid cell leukemia-1 (Mcl-1), and found that Mcl-1 was potentially targeted by miR-153. The forced expression of miR-153 inhibited the expression of Mcl-1 and luciferase activity, which was reversed by its antisense inhibitor. Furthermore, it was shown that the inhibition of miR-153 induced autophagy during oxidative stress, and that its effects of autophagy induction and apoptosis inhibition were efficiently abrogated by Mcl-1 small interfering RNA. In conclusion, the results of the present study elucidated a novel mechanism by which miR-153 regulates the survival of cardimyocytes during oxidative stress through the modulation of apoptosis and autophagy. These effects may be mediated directly by targeting Mcl-1. These finding revealed the potential clinical value of miR-153 in the treatment of cardiovascular disease.
The normal heart rhythm is particularly dependent upon proper contractile function of cardiomyocytes. However, the reactive oxygen species (ROS) generated under several pathological conditions, including ischemia-reperfusion injury, may be one of the major causes of heart muscle injury (
Previous studies of microRNAs (miRs) have indicated a a number of novel mechanisms of heart diseases. It has been reported that miRs not only control arrhythmogenesis, but are also significantly involved in the regulation of cardiomyocyte death (
As one of the major cascades of apoptosis, the intrinsic apoptotic pathway is initiated from mitochondria and is tightly controlled by B cell-lymphoma-2 (Bcl-2) family member proteins. One member of the Bcl-2 family, myeloid cell leukemia-1 (Mcl-1), has been reported to exert critical functions in apoptosis and mitochondrial homeostasis (
The present study aimed to identify a novel microRNA-associated mechanism in cardiac oxidative stress. A potential binding site of miR-153 was identified in the Mcl-1 3′-untranslated region, suggesting that miR-153 may be involved in this process. Therefore, multiple experimental methods were used to confirm our hypothesis that miR-153 regulates cardiomyocytes survival upon oxidative stimuli. The present study identified a novel role of miR-153 in regulating apoptosis and autophagy, and thus may provide certain clinical implications for the treatment of oxidative stress associated heart syndrome.
A primary culture of rat ventricular cardiomyocytes was used, as described previously (
A standard transfection procedure was used for delivering miR-153 or miR-153 antisense inhibitor into the cells. Lipofectamine 2000 tranfection reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used, according to the manufacturer's protocol for the kit. The Mcl-1 small interfering (si)RNA, miR-153 and miR-153 antisense inhibitor were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The final transfection concentrations for siRNA and miR were 150 nmol/l.
To assess the extent of cell death, an MTT method was used. Briefly, the cells were plated (25,000 cells/ml) in 96-well plates and treated, as indicated in each experiment. The cells were treated with 100
Total RNA was isolated from the cardiomyocytes using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Reverse transcription was performed using a specific stem-loop primer obtained from Guangzhou RiboBio Co., Ltd. SYBR green master mix (Promega Corp., Madison, WI, USA) was used to amplify the cDNA (1:15; 5
Western blot analysis was performed to detect the expression levels of the proteins of interest. Briefly, the cells were homogenized with SDS lysis buffer (Beyotime Institute of Biotechnology, Beijing, China) and denatured with sample buffer (Beyotime Institute of Biotechnology). The protein quantity was determined using a bicinchoninic acid assay. Electrophoresis of 30
A fragment of the Mcl-1 3′UTR was amplified by PCR, and the short sequence was inserted into the 3′UTR of an pMIRGLO construct, which expresses firefly luciferase. HEK293 cells (20,000/ml) were co-transfected with 0.1
All data are presented as the mean ± standard error of the mean. Comparisons between two groups were performed using Student's
The present study first examined the expression level of miR-153 under oxidative stress. The cardiomyocytes were treated with H2O2 for different time periods to induce apoptosis in the cardiomyocytes. Using western blot analysis, H2O2 was found to induce caspase-3 and 9 activation in a time-dependent manner (
Following the above observation that miR-153 was upregulated during oxidative stress, the present study subsequently transfected cells with the antisense inhibitor of miR-153 (anti-miR-153) to manipulate its endogenous level. Anti-miR-153 significantly reduced the expression of miR-153 under the H2O2 stimuli (
miRs commonly function to repress gene expression by binding to the 3′UTR region of mRNA. Using bioinformatical alignment, the present study identified a potential binding site within the 3′UTR of Mcl-1 (
As Mcl-1 has been established as a target of miR-153, and it has been previously shown to affect cardiac autophagy, the present study investigated whether Mcl-1-dependent autophagy is also under the control of miR-153 (
In the present study, it was demonstrated that miR-153 was involved in oxidative stress-induced cell death in cardiomyocytes. On investigating the molecular mechanism by which miR-153 regulated cell survival, the present study identified Mcl-1 as its direct target. As a consequence, the cardiomyocytes exhibited limited autophagic activity and enhanced apoptosis, which may have accounted, at least partially, for the H2O2-induced cardiomyocyte death. These results may have important clinical implications for the prevention and treatment of cardiac syndromes, including ischemia-reperfusion injury.
miRs have emerged as a class of important regulators in the heart. The interplay between miRs and several critical regulators in the apoptotic machinery have attracted wide attention in cardiac investigations. For example, a previous study by Wang
Apoptosis and autophagy are two essential biological processes, which act reciprocally to control cell survival under oxidative stress. It has been reported that basal autophagic activity is required for heart cells to maintain homeostasis, and that inhibition of this process by the deletion of its essential gene, ATG5, leads to heart failure (
The way in which miRs are regulated under different pathological conditions is an important issue and requires further understanding of the behavior of cardiomyocytes. Previous studies demonstrating the regulation of transcriptional factors of miRs may provide insights into this issue. For example, serum response factor transcriptionally regulates miR-1 in the heart, and p53 transcriptionally downregulates the expression of miR-499 to promote apoptosis (
In conclusion, the present study demonstrated the key role of miR-153 in oxidative stress-induced apoptosis. Mcl-1 was identified as a novel target, through which miR-153 regulated essential programs, including apoptosis and autophagy. These results may provide novel clues for understanding the control by miRs of multiple effects under certain stresses. The results of the present study may also guide the development of novel therapies for cardiac syndromes.
miR-153 is upregulated on exposure to apoptotic stimuli induced by oxidative stress. (A) Expression levels of cleaved-caspase-3 and cleaved-caspase-9 following H2O2 stimulation for various periods or time. Statistical analyses of the band densities of (B) cleaved caspase-3 and (C) cleaved caspase-9. (D) Cell viability of cardiomyocytes at each time point. (E) Relative expression of miR-153 at each time point. Data are presented as the mean ± standard error of the mean. *P<0.05, vs. control (n=5). miR, microRNA; O.D., optical density.
Inhibition of miR-153 reduces apoptosis of cardiomyocytes. (A) Relative expression of miR-153 following anti-miR-153 transfection. (B) Expression levels of cleaved-caspase-3 and cleaved-caspase-9 in cardiomyocytes subjected to different treatments. Statistical analyses of the band densities of (C) cleaved capsase-3 and (D) cleaved caspase-9. Cardiomyocytes were transfected with anti-miR-153 or anti-miR-NC for 48 h, and were then incubated with H2O2 for 24 h. (E) Cell viability of the transfected cardiomyocytes. Data are presented as the mean ± standard error of the mean. *P<0.05, vs. anti-miR-NC (n=5). miR, microRNA; anti-miR-53, miR-153 antisense inhibitor; NC, negative control; O.D., optical density.
Mcl-1 is a target of miR-153. (A) Predicted binding sites of rno- miR-153 and rat Mcl-1 3′UTR. (B) Enforced expression of miR-153 inhibited the expression of Mcl-1. (C) Statistical analysis of the band densities of Mcl-1. (D) miR-153 inhibited luciferase activity, whereas anti-miR-153 partially reversed this inhibition. Data are presented as the mean ± standard error of the mean. *P<0.05, vs. miR-NC; #P<0.05, vs. miR-153 (n=5). miR, microRNA; 3′UTR, 3′ untranslated region; Mcl-1, myeloid cell leukemia-1; NC, negative control.
miR-153 inhibits Mcl-1-dependent autophagy. (A) Expression levels of LC3 and Mcl-1 following transfection with anti-miR-153 or si-Mcl-1. Statistical analyses of band densities of (B) LC3-II and (C) Mcl-1. The cardiomyocytes were co-trasnfected with anti-miR-153 and si-Mcl-1 or their negative control strands for 48 h, as indicated, and were then incubated with H2O2 for 24 h. (D) Cell viability of the transfected cardiomyocytes. Data are presented as the mean ± standard error of the mean. *P<0.05, vs. anti-miR-NC+si-NC; #P<0.05, vs. anti-miR-153+si-NC (n=5). miR, microRNA; Mcl-1, myeloid cell leukemia-1; si, small interfering; NC, negative control; O.D., optical density.