miR-21/LncRNA GAS5介导抗凋亡在肾脏缺血/缺氧耐受中的作用机制研究

Ischemic acute kidney injury (AKI) is a common and critical disease. Cell apoptosis is an important pathophysiologic mechanism of ischemic AKI. Our study confirmed that miR-21 was involved in the regulation of cell apoptosis and contributed to the renal protection conferred by different pretreatment (ischemic preconditioning, Xenon and drugs). Long non-coding RNA (LncRNAs) can also be regulated by miRNAs as target genes. LncRNA GAS5 is a key regulator of apoptosis. Our pilot study has found that the effect of ischemia/reperfusion injury or ischemic preconditioning on the expression of renal GAS5 and different expression trend between miR-21 and GAS5 in hypoxic/reoxygenation cells. So we put forward the scientific assumptions: miR-21 might also protect the kidneys by inhibiting lncRNA GAS5 pathway. On the basis of the previous work, we intend to clarify the reciprocal relationship between miR-21 and GAS5, and further explore the protective function of miR-21/GAS5 in different ischemic/anoxic tolerance models and its underlying mechanism, from the molecules, cells, tissues and animal level, through inhibition or overexpression of GAS5 and miR-21, combined with RNA-pull down technology and miR-21 transgenic mouse. GAS5 will likely be a new target for the diagnosis and prevention of AKI.

缺血性急性肾损伤（AKI）是临床常见危重病症，细胞凋亡是其重要病理生理机制。我们研究已证实miR-21参与细胞凋亡的调控，在不同预处理（预缺血、氙气和药物）的肾脏保护中起重要作用。非编码RNA（LncRNAs）GAS5是细胞凋亡的关键调控因子。我们的预实验已发现缺血/再灌注或预缺血对肾脏GAS5表达的影响，且miR-21与GAS5的表达趋势相反。为此我们提出科学假设：miR-21可能通过GAS5途径，高效发挥肾脏保护作用。本项目拟在前期工作基础上，从分子、细胞、组织和整体动物水平层层深入研究，通过正负向调控GAS5和miR-21，结合RNA-pull down、miR-21转基因小鼠等技术，明确miR-21与GAS5的相互作用及机制，深入探讨miR-21/GAS5在不同方式诱导缺血/缺氧耐受中肾脏保护作用的新机制，而GAS5也可望成为AKI的防治新靶点。

缺血性急性肾损伤（AKI）是临床常见危重病症，细胞凋亡是其重要病理生理机制。我们研究已证实miR-21参与细胞凋亡的调控，在不同预处理的肾脏保护中起重要作用，并证实血小板反应蛋白（TSP-1）为miR-21下游凋亡相关的靶基因。非编码RNA（LncRNAs）GAS5是细胞凋亡的关键调控因子。根据既往研究，同样通过双侧肾蒂夹闭缺血35min后再灌注，成功构建肾脏缺血/再灌注（I/R）小鼠模型，I/R诱导肾脏GAS5表达，体外研究中随着细胞低氧时间的延长，GAS5表达逐渐增加，且表达高峰在低氧24h。进一步证实GAS5通过调控TSP-1促进细胞凋亡。进而我们观察到随着再灌注时间的延长，GAS5和TSP-1的表达显著上调，并在再灌注12小时时达到高峰，miR-21表达在再灌注48h内逐渐上升。利用LNA anti-miR-21体内抑制miR-21，削弱晚期缺血预适应（IPC）的肾脏保护作用，肾组织GAS5和TSP-1表达显著上调，伴有凋亡比例显著增加，由此说明IPC诱导的肾脏miR-21负向调控GAS5、TSP-1的表达发挥肾脏保护作用。此外，细胞缺氧24h复氧3h的条件下，通过GAS5 siRNA或质粒转染可反向调节miR-21和下调TSP-1的表达。同时荧光素酶报告基因检测技术验证miR-21对应的靶基因GAS5，进而先后利用miR-21 mimic/nc与质粒pcDNA-GAS5共转染、pcDNA-Vector/GAS5与miR-21 mimic共转染细胞， 证实GAS5作为 miR-21的“海绵”RNA，间接调控TSP-1，共同发挥抗细胞凋亡作用，减轻肾脏损伤。最后，我们利用氯化钴和丹参多酚酸诱导miR-21高表达，发挥肾脏保护作用。本项研究以miR-21/lncRNA GAS5为主线，阐明miR-21在肾脏缺血/缺氧耐受中保护作用的新机制，本研究不仅有望在AKI发病机制的理论方面获得创新和突破，为今后lncRNAs在肾脏疾病的研究奠定基础，并有可能为AKI的有效防治找到新的靶点和策略。