LncRNA-Gm26749作为ceRNA调控miR-196a-5p介导糖尿病肾病系膜细胞肥大的机制研究

Mesangial cell (MC) hypertrophy serves as an early key point responsible for the pathogenesis and of diabetic nephropathy (DN), which is regarded as central mechanism leading to loss of renal function. Long non-coding RNA (lncRNA) regulates microRNA ( miRNA)-mediated gene silence by binding miRNA response element (MRE), which play an important role in the pathogenesis of DN. However, the underlying mechanism remains unknown. Our preliminary data indicated that miR-196a-5p regulated high glucose-induced MC hypertrophy. LncRNA- Gm26749 upregulated remarkably in MCs in the presence of high glucose. And also the level of Gm26749 exihibits a reverse association with miR-196a-5p. Bioinformatics analysis indicated that Gm26749 shared a common MRE binding site with the target gene of miR-196a-5p. Based on a new competitive endogenous RNA (ceRNA) conception, we hypothesize that Gm26749 might act as a ceRNA involving in the pathogenesis of miR-196a-5p-mediated mesangial cell hypertrophy in diabetic nephropathy. To test our hypothesis, in this study in vitro we will investigate the role of Gm26749 in MC hypertrophy exposure to high glucose. Further more, we will deeply insight into the molecular mechanism, by which Gm26749 functions as a ceRNA involves in the pathogenesis of miR-196a-5p-mediated MC hypertrophy in diabetic nephropathy in vitro and in vivo. These results will contribute to identify the role and underlying mechanism of this novel signaling pathway of Gm26749-miR-196a-5p in the pathogenesis of MC hypertrophy of DN, which might provide theoretical basis of intervention at early stage of DN.

系膜细胞(MC)肥大是糖尿病肾病（DN）发病的关键始动环节，是肾功能丧失的中心机制；LncRNA通过MRE位点调控miRNA诱导的基因沉默，与DN密切相关，但机制尚不清楚。我们前期发现：miR-196a-5p调控高糖诱导的MC肥大，高糖诱导MC lncRNA Gm26749显著上调，与miR-196a-5p负相关，生物信息学预测Gm26749 与miR-196a-5p 靶基因存在相同MRE位点。结合ceRNA调控假说，我们推测Gm26749可能作为ceRNA调控miR-196a-5p介导的MC肥大。为此，本项目拟研究Gm26749在体外高糖诱导MC肥大中的作用；并结合体内、外实验探讨Gm26749作为ceRNA参与miR-196a-5p调控MC肥大的作用和分子机制，明确Gm26749-miR-196a-5p信号通路调控DN MC肥大的功能和作用途径，为DN早期干预提供实验理论依据。

糖尿病肾病（DN）已成为终末期肾病的重要原因，严重威胁患者的生命和健康；目前DN的治疗尚无特效药物；因此探索DN发病机制，寻找针对性防治措施具有重要现实意义。肾小球系膜细胞（MC）肥大是DN早期病理特征之一，并且在DN进展中发挥着重要作用。基于我们前期发现：miR-196a-5p调控高糖诱导的MC肥大，高糖诱导MC lncRNA Gm26749显著上调，与miR-196a-5p负相关；通过构建DN小鼠模型、高通量测序及生信分析，发现lncRNA Gm26749可能通过调控细胞周期循环、p53信号通路及PI3K-Akt信号通路介导DN发生发展；收集DN患者的血尿、组织样本及临床病例资料，采用qRT-PCR、原位杂交（ISH）等方法，验证了lncRNA Gm26749高表达于DN患者的肾组织和血清，相关性分析显示lncRNA Gm26749水平与尿蛋白水平成正相关，与eGFR水平成负相关。同时，采用体内外实验、数据库挖掘及生信分析，全方位的对糖尿病肾病非编码RNA进一步探究，发现miR-30b-5p、LINC00472及多个circRNA可能参与了糖尿病肾病的发生发展。利用空气流动辅助解吸电喷雾电离质谱成像(AFADESI-MSI)同时获得db/db和db/m小鼠肾脏的原位代谢谱，同时利用气相色谱-质谱联用技术(GC-MS)和超高效液相色谱-质谱联用技术(UPLC-MS)组成的综合代谢组学技术，检测肾脏均质物中代谢物的总量；在AFADESI-MSI原位检测中我们发现22种组织特异性的代谢紊乱，包括谷胱甘肽、牛磺酸、精胺、亚精胺、组胺及一系列脂肪酸；结合综合代谢组学分析，首次在2型DKD小鼠肾脏中同时发现并定位了5条功能失调的代谢途径，他们是牛磺酸代谢通路、精氨酸和脯氨酸代谢通路、组氨酸代谢通路、不饱和脂肪酸生物合成通路及脂肪酸降解途径；为DKD复杂代谢重编程的分子机制提供新的见解。此外，从表观遗传学的角度深度挖掘GEO数据库，并进行相应分析，发现核心调控基因的甲基化状态改变可能是DN发病的潜在因素。以上研究，为糖尿病肾病发病机制的阐明提供了理论依据，为临床诊疗提供了新思路。