Abstract
Background
Autophagy plays a significant role in myocardial ischemia-reperfusion (IR) injury. So it is important to inhibit autophagy to protect cardiomyocytes besides anti-apoptosis. MiRNA has been demonstrated to protect cardiomyocytes against apoptosis during IR, while whether it has anti-autophagy effect has not been known. The aim of this study was to investigate whether miR-204 regulated autophagy by regulating LC3-II protein, which is the marker of autophagosome during myocardial IR injury.
Methods
Adult SD rats were randomized to Control and IR groups. IR group was treated with 30 min ischemia by ligating the left anterior descending coronary artery, followed by 2 h reperfusion by loosing the ligation. The expression of miR-204 was measured by RT-PCR, and LC3 protein was measured by western-blot.
Results
We found that IR induced cardiomyocytes autophagy, together with down-regulation of miR-204 and up-regulation of LC3-II protein. And, we have found that LC3-II protein was regulated by miR-204, using the method of transferring miR-204 mimic or AMO-204 into the cardiomyocytes, before.
Conclusions
These studies provided evidence that miR-204 played an important role in regulating autophagy through LC3-IIprotein during IR.
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Background
Autophagy is a type of programmed cell death. It has been suggested to be essential for cell homeostasis [1–3]. It can determine the cell survival together with apoptosis and necrosis [4, 1.
Western-blotting
Protein concentration was determined with BCA protein assay kit according the manufacturer's protocol. Equal amounts of protein (60 ug) from the cardiomyocytes were subjected to Western-blotting analysis to evaluate LC3 expression with ECL detection kit (Amersham Biosciences, Piscataway, NJ). LC3 immunoreactivity was detected using a rabbit antiserum specific for rat Beclin1 protein (Sigma, USA) as primary antibodies. Detection of antigen-antibody complex formation was performed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody. The LC3 concentrations were normalized with β-actin.
Statistical analysis
Quantitative data are presented as mean ± standard error. Statistical significance was determined using T test or one-way ANOVA. P < 0.05 was considered statistically significant.
Results
Myocardium injury was induced by IR
The extent of myocardial infarction was evaluated after reperfusion. Representative photographs of midventricular cross sections of evans blue and TTC-stained hearts were taken from Control and IR groups. INF/AAR and LDH were shown in Figure 1.
IR decreased the expression of miR-204
To demonstrate the effect of IR on miR-204, we compared the miR-204 between the control group and IR group (n = 10). It was found that IR significantly decreased miR-204 with the method of Real-time PCR. (Figure 2)
IR up-regulated the protein level of LC3-II
As a marker of autophagosome, the protein level of LC3-II represents the amount of autophagosome. So we compared the ratio of LC3-II/LC3-I between the control group and IR group (n = 10), and found that it was enhanced by IR. (Figure 3)
Discussion
In recent years, autophagy has attracted great interest because it was involved in many physiological processes. If autophagy destroys the cytosol and organelles beyond a certain threshold, autophagic cell death will occur [25]. Autophagy was detrimental during reperfusion although it protected the cardiomyocytes during ischemia, which has been demonstrated by Matsui and his collaborators [26]. It has been reported that autophagy contributed to cell death when apoptosis is inhibited, and sometimes the early stages of autophagy were required for apoptosis [24]. LC3-II was the marker of autophagosome, instead of LC3-I. So the ratio of LC3-II/LC3-I could stand for the level of autophagy. In our study, we found that IR up-regulated the protein expression of LC3-II together with increasing the ratio of autophagy cell. So it would be beneficial for the revascularized hearts, to find a method of regulating LC3-II expression.
MiRNA is a group of small, non-coding RNAs which regulates gene expression in a sequence-dependent manner. They are endogenous regulators of gene expression, and have been demonstrated to be involved in cardiac IR injury. According to the bioinformatics of Targetscan, miR-204, which has anti-apoptosis effect [23], may regulate the expression of LC3-II.
In our study, it was found that IR could down-regulate miR-204 together with up-regulated LC3-II protein. When miR-204 mimic was transferred into cardiomyocytes, LC3-II protein was attenuated, and LC3-II protein was up-regulated by AMO-204 which was concentration- dependented as other miRNAs [27]. But LC3-I was not regulated by miR-204, as the previous study [28]. These results demonstrated that miR-204 may regulate cardiomyocytes autophagy through LC3-II during IR injury.
Conclusions
Our results demonstrated that miR-204 played an important role by regulating LC3-II protein during IR. So it became possible to control the autophagy under a beneficial threshold by regulating miR-204 expression, for protecting the cardiomyocyte against IR injury. But it is still unknown how did IR regulated the expression of miR-204, and this will be our next work.
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Acknowledgements
This work was supported by the National Nature Science Foundation of China (No.30901470, No.30800375 and No.30700157).
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Authors' contributions
Jian **ao carried out the IR model, RT-PCR and Western-blot studies, and drafted the manuscript. **aoyan Zhu carried out the miR-204 transferring into cardiomyocytes, and participated in the sequence alignment of miR-204 mimic and AMO-204. Bin He carried out the primers synthesis of miR-204 and participated in drafting the manuscript. Bo Kang carried out the cardiomyocytes culture. Yufeng Zhang carried out the LDH assay. Zhinong Wang and **n Ni conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Jian **ao, **aoyan Zhu, Bin He contributed equally to this work.
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**ao, J., Zhu, X., He, B. et al. MiR-204 regulates cardiomyocyte autophagy induced by ischemia-reperfusion through LC3-II. J Biomed Sci 18, 35 (2011). https://doi.org/10.1186/1423-0127-18-35
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DOI: https://doi.org/10.1186/1423-0127-18-35