miR-21对糖尿病肾小管上皮细胞SnoN蛋白表达的调控作用及其机制研究

The incidence of diabetic nephropathy (DN) continues to rise. The main pathology of DN is characteristic of renal tubular fibrosis. There is no effective methods to treat DN up to now. SnoN is an important negative regulator of TGF-β1/Smad signaling pathways that plays a key role in the organ fibrosis . MiR-21 can promote the renal fibrosis and bioinformatic analysis show SnoN is its potential target gene. Our recent studies have shown that SnoN protein level reduced and mirR-21 level rised in renal tubular epithelial cells(RTECs) which may be an important mechanism of renal fibrosis in DN and that SnoN expression change occurred at post-transcription level.But we did not know the relationship between SnoN and miR-21 through consulting relevant literatures. Therefore we presume that the SnoN protein reduction in DN is related to miR-21. We intend to employ diabetes mellitus rats model and RETCs under the condition of high concentrations glucose as the research object and to use the methods such as molecular biology, which enhance or inhibit the miR -21 expression level in RETCs.Then we observe cell phenotype and SnoN mRNA and SnoN protein change in RETCs under the condition of high glucose. Later ,we use dual luciferase report gene method to test whether SnoN is the target gene of miR -21.We will discuss the promoting effect of miR -21 on renal fibrosis of DN and its regulatory function of SnoN expression and the molecular mechanism ，so as to offer evidences seeking novel targets and methods for the effective prevention and treatment of DN.

糖尿病肾病（DN）发病率逐年增高，随病情进展发生肾纤维化，发病机制复杂，缺乏有效治疗措施。我们前期发现，DN肾小管上皮细胞（RETC）SnoN蛋白减少可能是致肾纤维化的重要机制，且SnoN表达减少发生于转录后，但其调控机制尚未明确；我们亦发现DN发病过程中RETC的miR-21显著增高，而生物信息学分析显示SnoN是miR-21的候选靶基因，故推测RETC中miR-21增多可能与SnoN蛋白减少有关，然而SnoN与miR-21之间的关系尚未见研究报道。本研究拟以糖尿病大鼠和高糖培养的RETC为研究对象，采用分子生物学等方法，动态观察SnoN和miR-21表达变化、上调或下调RETC中miR-21后对SnoN蛋白表达的影响，用双荧光素酶报告基因法验证SnoN是否为miR-21的靶基因。从体内和体外水平探讨miR-21对糖尿病RETC中SnoN表达的调控作用及其分子机制，为DN防治提供新思路。

糖尿病肾病(DN) 是糖尿病最严重的微血管并发症，是导致终末期肾病致患者死亡的主要原因，随着全球DN 发病率也呈逐年增长趋势，其发病机制复杂且缺乏有效治疗措施。SnoN作为TGF-β1/Smads信号通路负调节因子，其蛋白表达减少可能DN肾纤维化的关键致病机制，但SnoN在DN病程中的表达调控机制尚不清楚。DN大鼠及体外高糖处理的肾小管上皮细胞（RTECs）中，miR-21明显上调，而生物信息学分析表明SnoN可能是miR-21的靶基因。故本研究以DN大鼠模型和体外高糖培养的RTECs为研究对象，采用病理学、免疫学、生物化学、分子生物学及细胞生物学等技术，分析 miR-21、SnoN以及相关指标的表达变化，通过构建miR-21表达载体和抑制载体分别转染 RTECs，分析过表达或敲低miR-21后RTECs中 SnoN表达变化，构建重组的荧光素酶报告基因质粒，进一步验证SnoN是否为miR-21的靶基因，利用SnoN基因敲低及基因转染技术证实SnoN是否可以影响miR-21表达等，探讨miR-21对RTECs中SnoN蛋白表达的调控作用及其分子机制。结果表明：在高糖环境下，TGF-β1通过激活RTECs中其下游的Smad3信号通路，上调miR-21的表达，增多的miR-21可能通过抑制SnoN mRNA的翻译，导致SnoN蛋白水平降低，使得TGF-β1/Smad3信号通路失去控制而过度活化，促进DN的发生发展；高糖环境下，RTECs过表达miR-21可促进上皮细胞向间充质细胞转分化（EMT）发生和细胞外基质（ECM）沉积，而miR-21抑制可减轻EMT的发生和ECM的沉积，miR-21可能通过调控SnoN表达，发挥致纤维化效应；高糖环境下，使RTECs过表达SnoN可抑制miR-21表达、EMT和纤维化病变；相反，敲低SnoN可以促进miR-21的表达、EMT和纤维化病变。本研究还从泛素化降解方面寻找DN发病过程中调控RTECs中SnoN蛋白表达的可能因子和机制，发现BMP-7、氧化苦参碱、抗氧化剂α-硫辛酸及Akadia等均可通过不同机制上调SnoN 蛋白，抑制TGF-β1/Smads信号通路，而减轻或延缓DN肾纤维化。综上，本项目系统研究DN进程中miR-21的作用，与SnoN的关系及可能调控机制，为DN防治药物的开发提供新的思路和靶点。