Wei Qingqing, Liu Yong, Liu Pengyuan, Hao Jielu, Liang Mingyu, Mi Qing-Sheng, Chen Jian-Kang, Dong Zheng
Department of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia;
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin;
J Am Soc Nephrol. 2016 Sep;27(9):2784-96. doi: 10.1681/ASN.2015080870. Epub 2016 Mar 14.
MicroRNAs have been implicated in ischemic AKI. However, the specific microRNA species that regulates ischemic kidney injury remains unidentified. Our previous microarray analysis revealed microRNA-489 induction in kidneys of mice subjected to renal ischemia-reperfusion. In this study, we verified the induction of microRNA-489 during ischemic AKI in mice and further examined the underlying mechanisms. Hypoxia-inducible factor-1α deficiency associated with diminished microRNA-489 induction in cultured rat proximal tubular cells subjected to hypoxia and kidney tissues of mice after renal ischemia-reperfusion injury. Moreover, genomic analysis revealed that microRNA-489 is intronic in the calcitonin receptor gene, and chromatin immunoprecipitation assays showed increased binding of hypoxia-inducible factor-1 to a specific site in the calcitonin receptor gene promoter after hypoxia. Inhibition of microRNA-489 increased apoptosis in renal tubular cells after ATP depletion injury in vitro, whereas microRNA-489 mimics mediated protection. In mice, inhibition of microRNA-489 enhanced tubular cell death and ischemic AKI without significantly affecting tubular cell proliferation. Deep sequencing identified 417 mRNAs that were recruited to the RNA-induced silencing complex by microRNA-489. Of the identified mRNAs, 127 contain microRNA-489 targeting sites, and of those, 18 are involved in the cellular stress response, including the poly(ADP-ribose) polymerase 1 gene implicated in ischemic kidney injury. Sequence analysis and in vitro studies validated poly(ADP-ribose) polymerase 1 as a microRNA-489 target. Together, these results suggest that microRNA-489 is induced via hypoxia-inducible factor-1 during ischemic AKI to protect kidneys by targeting relevant genes.
微小RNA与缺血性急性肾损伤有关。然而，调节缺血性肾损伤的特定微小RNA种类仍未明确。我们之前的微阵列分析显示，在经历肾缺血再灌注的小鼠肾脏中，微小RNA-489被诱导表达。在本研究中，我们验证了小鼠缺血性急性肾损伤期间微小RNA-489的诱导情况，并进一步研究了其潜在机制。缺氧诱导因子-1α缺乏与缺氧培养的大鼠近端肾小管细胞以及肾缺血再灌注损伤后小鼠肾脏组织中微小RNA-489诱导的减少有关。此外，基因组分析表明，微小RNA-489位于降钙素受体基因的内含子中，染色质免疫沉淀分析显示，缺氧后缺氧诱导因子-1与降钙素受体基因启动子中的特定位点结合增加。抑制微小RNA-489可增加体外ATP耗竭损伤后肾小管细胞的凋亡，而微小RNA-489模拟物则介导保护作用。在小鼠中，抑制微小RNA-489可增强肾小管细胞死亡和缺血性急性肾损伤，而对肾小管细胞增殖无显著影响。深度测序鉴定出417种被微小RNA-489招募到RNA诱导沉默复合体的mRNA。在鉴定出的mRNA中，127种含有微小RNA-489靶向位点，其中18种参与细胞应激反应，包括与缺血性肾损伤有关的聚（ADP-核糖）聚合酶1基因。序列分析和体外研究验证了聚（ADP-核糖）聚合酶1是微小RNA-489的靶标。总之，这些结果表明，在缺血性急性肾损伤期间，微小RNA-489通过缺氧诱导因子-1被诱导表达，通过靶向相关基因来保护肾脏。
