基于LncRNA-miRNA-mRNA共表达调控网络调节足细胞损伤探讨小檗碱治疗糖尿病肾病的作用机制研究

Diabetic nephropathy is one of the main complications of diabetic microangiopathy, and its pathogenesis is still uncertain for the time being. Our previous studies show that berberine could alleviate the podocyte injury, thus preventing the progression of diabetic nephropathy. In vitro study, we find that there are significant differences in the miRNAs expression after podocyte injury (such as miR193b-5p and miR200b, etc.), and some of which can be affected by berberine. In addition, we also discover that the alterations of corresponding upstream LncRNA, downstream mRNA genes and some signaling pathways, which are closely related to the miRNA193b-5p expression. However, the precise mechanisms remain elusive. On the basis of previous important discovery, we are going to explore and verify the target LncRNA and miRNA in cultured podocyte and diabetic nephropathy model rat, then clarify the regulatory molecular mechanism of LncRNAGm15406-miR193b-5p-mRNA co-expressed regulatory network on podocyte injury. With the further study, the alleviating actions and the precise molecule mechanisms of above RNAs and berberine on podocyte injury and diabetic nephropathy progression will be comprehensively and accurately revealed through regulating LncRNAGm15406, miR193b-5p and the corresponding mRNA gene respectively. Our study will lay a new theoretical foundation for the pathogenesis of diabetic nephropathy and provide a new strategy for the diabetic nephropathy treatment.

糖尿病肾病是糖尿病微血管病变的主要并发症之一，其发病机制至今尚未阐明。我们前期研究发现小檗碱可通过缓解足细胞损伤抑制糖尿病肾病的发展；足细胞损伤前后有多种miRNA(如miR193b-5p、miR200b等)表达存在显著差异，其中小檗碱可影响部分miRNA的表达；同时，足细胞中与miRNA表达密切相关的LncRNA、mRNA及信号分子均发生变化，然而具体机制不明。本项目拟以足细胞和大鼠模型为研究对象，确立LncRNA与miRNA靶点；以诱导过表达的miR193b-5p为重点，阐明LncRNAGm15406-miR193b-5p-mRNA共表达调控网络对足细胞损伤的调节机制；分别调控LncRNAGm15406、miR193b-5p及mRNA，揭示上述RNA与小檗碱缓解足细胞损伤及抑制糖尿病肾病作用的分子机制。本研究将为糖尿病肾病的发病机制提供新的理论依据，为糖尿病肾病的药物治疗提供新的思路。

糖尿病肾病(DN)是糖尿病最常见且最严重的微血管并发症之一，也是终末期肾病的主要诱因，但其发病机制尚未阐明。非编码RNA(ncRNA)作为主要的表观遗传机制通过调节多种基因的表达在包括DN在内的多种疾病中发挥着不可忽视的作用。与此同时，课题组前期研究发现多种ncRNA在DN引发的足细胞损伤和凋亡过程中异常表达，但具体机制不明。因此，深入探索DN的发病机制、探讨ncRNA在DN足细胞损伤中的作用与相关机制并以此为基础探寻潜在的干预治疗靶点与药物将为足细胞损伤与DN的治疗提供重要的参考。基于此，本研究建立2型DN小鼠模型(T2DN)并给予不同剂量的小檗碱治疗发现生化指标(如血糖、血清肌酐和尿总蛋白等)显著升高、肾脏发生病理改变(肾小球硬化、基底膜增厚、糖原沉积等)。与此同时，足细胞黏附能力降低、足突的融合、消失和足细胞的损伤与凋亡等现象增加，而小檗碱可一定程度的逆转上述异常变化。高糖(30mmol/L)刺激足细胞在引起损伤与凋亡的同时葡萄糖转运蛋白(Glucose transporter 1, GLUT1)表达显著增加。全转录组测序与CNC关联分析发现LncRNA Gm12866与葡萄糖转运蛋白(GLUT1)密切相关。抑制GLUT1可降低葡萄糖摄入，减少足细胞损伤与凋亡，增强足细胞活性，这表明高糖状态下干预GLUT1可减少GLUT1的膜表达与转位，减少葡萄糖过度摄取，进而减轻足细胞损伤与凋亡。生信分析发现Gm12866对GLUT1存在顺式调控可能，同时LncR Gm12866 FISH探针也表明LncR Gm12866主要表达于细胞核中。随后沉默LncR Gm12866发现GLUT1的mRNA与膜蛋白表达均存在一定程度的降低，足细胞对葡萄糖摄取减少，足细胞损伤与凋亡情况得到缓解。尾静脉注射慢病毒介导的LncR Gm12866显示其可一定程度的缓解前述生化指标异常与病理改变以及足细胞损伤等变化。综上所述，LncR Gm12866主要通过顺式调控GLUT1 mRNA，减少GLUT1的膜位移，进而影响葡萄糖的摄取与足细胞的损伤达到缓解DN的作用，小檗碱可通过影响GLUT1介导的糖转运影响DN的进展，成为潜在的DN治疗药物。