Cardiac Hypertrophy Laboratory, Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, Tamilnadu, India.
Cardiac Hypertrophy Laboratory, Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, Tamilnadu, India.
Exp Cell Res. 2018 Apr 1;365(1):46-56. doi: 10.1016/j.yexcr.2018.02.016. Epub 2018 Feb 23.
The physiological cardiac hypertrophy is an adaptive condition without myocyte cell death, while pathological hypertrophy is a maladaptive condition associated with myocyte cell death. This study explores the miRNome of α-2M-induced physiologically hypertrophied cardiomyocytes and the role of miRNA-99 family during cardiac hypertrophy. Physiological and pathological cardiac hypertrophy was induced in H9c2 cardiomyoblast cell lines using α-2M and isoproterenol respectively. Total RNA isolation and small RNA sequencing were executed for physiological hypertrophy model. The differentially expressed miRNAs and its target mRNAs were validated in animal models. Transcription factor binding sites were predicted in the promoter of specific miRNAs and validated by ChIP-PCR. Subsequently, the selected miRNA was functionally characterized by overexpression and silencing. The effects of silencing of upstream regulator and downstream target gene were studied. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during hypertrophy, of which miR-99 family was highly downregulated upon α-2M treatment. However, this miR-99 family expression was upregulated during pathological hypertrophy and confirmed in animal models. ChIP-PCR confirms the binding of Egr-1 transcription factor to the miR-99 promoter. Further, silencing of Egr-1 decreased the expression of miR-99. The overexpression or silencing of miR-99 diverges the physiological hypertrophy to pathological hypertrophy and vice versa by regulating Akt-1 pathway. Silencing of Akt-1 replicates the effect of overexpression of miR-99.
The results proved Egr-1 mediated regulation of miR-99 family that plays a key role in determining the fate of cardiac hypertrophy by regulating Akt-1 signaling.
生理心脏肥大是一种适应性状态，没有心肌细胞死亡，而病理性肥大是一种与心肌细胞死亡相关的失调状态。本研究探讨了α-2M 诱导的生理性肥大心肌细胞的 microRNA 组和 miRNA-99 家族在心脏肥大中的作用。分别使用α-2M 和异丙肾上腺素在 H9c2 心肌细胞系中诱导生理性和病理性肥大。对生理肥大模型进行总 RNA 分离和小 RNA 测序。在动物模型中验证差异表达的 miRNA 及其靶 mRNA。在特定 miRNA 的启动子中预测转录因子结合位点，并通过 ChIP-PCR 验证。随后，通过过表达和沉默对选定的 miRNA 进行功能表征。研究了上游调节剂和下游靶基因沉默的影响。对小 RNA 读数的分析揭示了肥大过程中大量 miRNA 的差异表达，其中 miR-99 家族在 α-2M 处理后高度下调。然而，在病理性肥大期间，miR-99 家族的表达上调，并在动物模型中得到证实。ChIP-PCR 证实了 Egr-1 转录因子与 miR-99 启动子的结合。进一步，沉默 Egr-1 降低了 miR-99 的表达。miR-99 的过表达或沉默通过调节 Akt-1 通路使生理性肥大向病理性肥大转变，反之亦然。沉默 Akt-1 复制了过表达 miR-99 的效果。
结果证明了 Egr-1 介导的 miR-99 家族的调节，通过调节 Akt-1 信号在决定心脏肥大命运方面起着关键作用。
