lncRNA AK154753在缺血再灌注急性肾损伤细胞凋亡中的作用及机制研究

Acute renal injury (AKI) is a complex clinical syndrome which varies different causes and is poor of effective treatment. Acute renal ischemia-reperfusion injury (IRI) is a common type of AKI. LncRNA refers to a class of non-coding RNA transcripts with a length greater than 200 nt, which is highly heterogeneous in structure and function. Studies have shown that specific lncRNA regulates apoptosis in heart and brain ischemia and reperfusion (I / R). However, there are few articles reported at home and abroad how lncRNA specifically regulates apoptosis in IRI. By LncRNA, MicroRNA chip and Realtime PCR, our pre-tests demonstrate that the expression of lncRNA AK154753, BAK and BCL2L11 were up-regulated in I / R kidney tissue while MIR-345-3p and MIR-708-5p were down-regulated， suggesting that lncRNA AK154753 might involve in the development of AKI induced by acute ischemia-reperfusion by regulating the pathway of MIR-345-3p/MIR-708-5p——BAK/BCL2L11. Therefore,the purpose of this study is to explore the role of apoptosis and molecular mechanisms of lncRNA AK154753 in Acute renal ischemia-reperfusion injury, in vivo and in vitro, around the regulatory network of "lncRNA AK154753——MIR-345-3p / MIR-708-5p——BAK / BCL2L11" ，so as to provide a new theoretical basis for the prevention and treatment of AKI.

急性肾损伤是一组复杂的临床综合征，病因多样，疗效欠佳。lncRNA是指一类长度大于200nt的非编码RNA转录本，其结构及功能具有高度异质性。研究表明不同的lncRNA在心、肝、脑等器官缺血再灌注损伤中对凋亡有调控作用。但是目前lncRNA是否参与急性缺血再灌注肾损伤的凋亡调控国内外鲜有报道。我们预实验已证实缺血再灌注小鼠肾组织lncRNA AK154753、BAK、BCL2L11表达上调，而MIR-345-3p、MIR-708-5p则表达下调，推测lncRNA AK154753可能通过影响MIR-345-3p、MIR-708-5p、BAK1、BCL2-L11表达调控细胞凋亡参与急性缺血再灌注肾损伤过程。本研究拟从体内、体外两个层次，进一步探讨上述调控网络，阐明lncRNA AK154753在急性缺血再灌注肾损伤细胞凋亡中的作用及分子机制，从而为防治AKI提供新的理论依据和新的治疗靶点。

急性肾损伤（Acute kidney injury, AKI）是一组复杂的临床综合征，病因多样，疗效欠佳。lncRNA是指一类长度大于200核苷酸的非编码RNA，调控基因表达。本项目拟探讨lncRNA AK154753 在缺血性AKI肾小管上皮细胞凋亡中的作用和机制。本项目的主要发现：1）对缺血性AKI 小鼠和假手术小鼠的肾组织进行全基因组表达谱分析，发现了差异表达的非编码RNA(ncRNA)和mRNA；生物信息学及RT-PCR分析证实相对假手术小鼠，缺血性AKI小鼠肾组织中lncRNA AK154753表达上调，miR-345-3p和miR-708-5p表达下调。2）体内发现相对假手术小鼠，缺血性AKI小鼠肾组织中促细胞凋亡蛋白Bak和Bim显著增加，TUNEL染色阳性的细胞增加；体外，流式细胞分析显示缺氧-复氧（模拟体内缺血再灌注）促进肾小管上皮细胞凋亡。3）荧光原位杂交发现lncRNA AK154753主要分布在肾小管上皮细胞的细胞质。4）双荧光素酶报告基因检测提示lncRNA AK154753与miR-345-3p、lncRNA AK154753与miR-708-5p、miR-345-3p与Bak、miR-708-5p与Bim间均存在相互作用。5）体外，过表达lncRNA AK154753下调miR-708-5p和miR-345-3p表达；利用miRNA mimic或inhibitor进一步分析发现miR-345-3p和miR-708-5p可分别调控Bak或Bim的表达。6）抑制lncRNA AK154753增加miR-345-3p和miR-708-5p的水平，并减轻缺氧-复氧诱导的细胞凋亡；抑制miR-345-3p或miR-708-5p的功能阻断lncRNA AK154753沉默引起的细胞保护，并增加Bak和Bim表达。综上所述，本项目的研究证实缺血再灌注诱导肾小管上皮细胞lncRNA AK154753表达，lncRNA AK154753表达增加降低细胞内miR-345-3p和miR-708-5水平，进而促进细胞凋亡蛋白Bak和Bim的表达和细胞凋亡。本项目的研究发现为以lncRNA AK154753作为AKI的防治靶点提供了实验依据。至今，本项目已发表期刊论文3篇，培养硕士研究生2人，部分研究结果整理待发表。