mTOR-自噬参与特定miRNA介导的硫化氢心肌保护作用的研究

Many studies has revealed that autophagy plays a key role in myocardial IR injury and can be regulated by mammalian target of rapamycin (mTOR), which is crucial in atuophagy regualtion. Our preliminary data demonstrated that hydrogen sulfide (H2S) down-regulated the expression of autophagy-related gene (Atg) and up-regulated the level of p-mTOR protein. Moreover, by using miRNA chip, it has been provided evidence that miRNA (miR-199a, miR-204 and miR-30) played important roles in myocardial IR injury and these miRNA could be regulated by H2S. According to bioinformation, the target genes of these miRNA included multiple Atgs and mTOR subunit. We hypothesised that H2S could inhibit autophage by modulating the specific miRNA through regulating mTOR-autophagy pathway. This study is designed to investigate whether specific miRNA could regulate mTOR-autophagy associated targert genes and how H2S could inhibit autophagy through miRNA pathway by modulating the specific miRNA.The proposed study will significantly advance our current understanding of the mechanisms of H2S regulating mTOR-Atg signaling and inhibiting myocardial autophagy. Results gained from this study will provide new vision and help with the development of novel therapies for clinical cardioprotection. Thus, this proposed study has theoretically significance and potentially practical value.

心肌细胞自噬在心肌缺血再灌注（IR）损伤中扮演重要角色。在自噬的调控机制中，雷帕霉素靶蛋白（mTOR）可能起关键作用。我们前期研究发现硫化氢可下调IR心肌细胞自噬相关基因（Atg）的表达，并上调p-mTOR蛋白。此外应用miRNA芯片在IR和对照组之间筛选出差异显著且心肌特异表达的miRNA（miR-199a、miR-204和miR-30）。生物信息学显示上述miRNA的靶基因包括多种Atg及mTOR亚单位。初步研究表明部分miRNA可被硫化氢调控，提示硫化氢可能通过miRNA，调控mTOR-自噬通路。本项目将以mTOR-自噬为切入点，通过上调/下调上述miRNA，观察其对自噬相关靶基因的调控，明确硫化氢如何通过调控miRNA干预mTOR-自噬通路，减少心肌细胞自噬发生。本研究有助阐明硫化氢调控mTOR-Atg表达、抑制心肌细胞自噬的可能机制，为硫化氢相关药物研发及临床心肌保护提供新思路。

心肌细胞自噬在心肌缺血再灌注（ischemia reperfusion，IR）损伤中扮演重要角色。适度的自噬是细胞完成自身代谢和细胞器更新的重要方式；而非生理性的、过度的自噬则会导致细胞过多死亡。自噬的发生受到Atg5、Beclin1、LC3等自噬相关基因（autophagy related gene，Atg）的调控。此外，在自噬的调控机制中，雷帕霉素靶蛋白（mTOR）发挥关键作用。.大量研究表明，心肌高表达或特异表达的miRNA参与调控了多种心脏生理和病理过程，特别在心肌IR损伤中扮演着重要角色。生物信息学研究发现部分miRNA可以调控Atg家族及mTOR表达。.本项目以mTOR-自噬为切入点，通过上调/下调特定miRNA，观察其对自噬相关靶基因的调控，明确硫化氢如何通过调控miRNA干预mTOR—自噬通路，减少心肌细胞自噬发生。.主要研究内容：观察H2S对再灌注性心肌细胞特定miRNA和mTOR—Atg表达的影响，研究H2S能否通过调控心肌高表达或特异表达的特定miRNA，干预mTOR—Atg的表达，减少IR心肌细胞自噬的发生，发挥心肌保护作用。.结果发现：（1）自噬抑制剂3-MA显著减轻心肌细胞IR损伤，提高作用。（2）H2S下调自噬相关基因表达，抑制自噬发生，从而减轻心肌细胞IR损伤，并且具有一定的浓度依赖性，浓度在30uM时效果最佳。（3）mTOR参与H2S调控IR心肌细胞自噬的作用。（4）miRNA参与调控心肌IR损伤，并受到H2S的调控。（5）特定miRNA调控自噬相关靶基因表达。.综上所述，本课题证实了H2S通过调控miRNA-TOR-自噬减轻再灌注性心肌细胞损伤，发挥心肌保护作用。.本研究有助阐明硫化氢调控mTOR-Atg表达、抑制心肌细胞自噬的可能机制，为H2S相关药物研发及临床心肌保护提供新思路。