LncRNA MALAT1调控高糖引发的肾小管上皮细胞凋亡的机制研究

Renal tubular injury plays a vital role in development of diabetic nephropathy (DN)，lncRNA MALAT1 interacting with function protein plays an important role in the pathological process of DN. Our pilot experimental results show that lncRNA MALAT1 increases in the renal tubules of DN rats, however, the mechanism underling MALAT1 affecting DN remains to be further discussed。Our preliminary study has shown that decreased SIRT1 deacetylase activity contributes to high glucose induced tubule epithelial cell injury. Therefore, we put forward the hypothesis that MALAT1 may contributes to the renal tubular epithelial cells apoptosis via regulating SIRT1 signaling pathway. To verify this hypothesis we will establish in vivo rats STZ diabetic kidney damage model and in vitro high glucose cultured renal tubular epithelial cells model, incorporating with ChIP, chromosome immunoprecipitation combined with quantitative PCR, cell transfection, the signaling pathway inhibition, aiming to explore expressional regulation of SIRT1 by abnormal expression of MALAT1 in DN renal tubular cells apoptosis and to indicate the mechanism of renal tubular apoptosis by MALAT1 in DN . This study will apply a new viewpoint of the lncRNA-MALAT1 to reveal the mechanism of renal tubular damage and provide a new method for prevention and control of diabetic nephropathy.

肾小管损伤在糖尿病肾病（DN）的发生发展中起重要作用，lncRNA MALAT1通过对功能蛋白的表达调控参与了DN的病理进程。我们的预实验结果显示，MALAT1在DN大鼠肾小管中表达升高，但作用机制有待进一步探讨。本课题前期研究显示SIRT1的去乙酰化活性下降在高糖下肾小管上皮细胞损伤中有重要调控作用。为此，我们提出假说，MALAT1可能通过影响SIRT1信号通路，参与高糖引发的肾小管上皮细胞凋亡。为验证这一假说，我们将建立DN大鼠模型和体外肾小管上皮细胞高糖培养模型，借助ChIP、染色体免疫共沉淀联合定量 PCR、细胞转染、信号通路阻断等技术探讨MALAT1调控SIRT1活性在DN肾小管上皮细胞凋亡中的作用，明确MALAT1通过肾小管凋亡促进DN发生的机制。本研究将从lncRNA MALAT1这个新视点为揭示DN肾小管损伤的发生机制奠定基础，为糖尿病肾病的防治提供新的思路。

糖尿病肾病 (Diabetic nephropathy, DN) 是糖尿病最严重的并发症之一。DN发病机制复杂，其中肾小管损伤是DN重要的病理特征之一。肾小管间质病变程度反映疾病的进展，且与肾功能损伤存在明显的相关性。近期研究证明，长链非编码RNA（long non-coding RNA，lncRNA）在糖尿病肾病中发挥重要作用。本课题组前期研究发现在糖尿病模型小鼠肾脏组织中LncRNA MALAT1表达上调，SIRT1表达下调。有研究发现高糖环境抑制SIRT1去乙酰化活性，引起肾小管上皮细胞的损伤。此外，Foxo1在糖尿病肾病大鼠肾皮质的活性降低，抑制足细胞凋亡，但对肾小管损伤的影响尚未报道。通过生物信息软件预测到lncRNA MALAT1可能与Foxo1存在结合位点，Foxo1可能与SIRT1启动子区存在结合位点。这提示LncRNA MALAT1可能会与Foxo1和SIRT1共同参与肾小管损伤。.研究内容.1.构建糖尿病小鼠模型，检测糖尿病小鼠与正常小鼠中MALAT1、SIRT1和Foxo1的表达。.2.高糖处理肾小管上皮细胞HK-2，检测MALAT1和SIRT1的表达变化。.3.高糖处理HK-2细胞并敲低MALAT1的表达，检测Foxo1和SIRT1的表达。.4.RNA免疫沉淀和RNA pull-down检测正常HK-2细胞和高糖诱导的HK-2细胞中，MALAT1和Foxo1是否结合。.5.CHIP实验检测正常HK-2细胞或高糖诱导的HK-2细胞或高糖诱导的MALAT1敲低的HK-2细胞中Foxo1和SIRT1启动子之间是否结合。.重要结果.1.糖尿病小鼠肾组织中MALAT1表达上调，而SIRT1表达下调。.2.高糖处理HK-2细胞后，MALAT1表达上调，而SIRT1表达下调。此外，MALAT1敲低可以逆转高糖诱导的SIRT1表达下调。.3.HK-2中MALAT1与Foxo1结合，且高糖促进了MALAT1和Foxo1的结合。.4.HK-2中Foxo1与SIRT1结合，且高糖抑制了Foxo1和SIRT1之间的结合，但是MALAT1的低表达逆转了这个结果..关键数据及科学意义.糖尿病小鼠肾组织和高糖处理的HK-2细胞中MALAT1表达上调，且MALAT1的干扰逆转了由高糖引起的细胞活力降低。说明MALAT1可作为治疗糖尿病中肾小管损伤中的潜在靶标。