lncGm2115-miR-150效应体介导Cxcr4/PI3K/Akt通路调控糖尿病肾病干细胞归巢修复的作用机制

Low homing ability of mesenchymal stem cells (MSCs) is challenging the celluar therapy of diabetic nephropathy (DN). However, the mechanism is not fully clear. Our previous studies revealed that miRNA participated in DN (NO.30800534), and miR-150 knockdown could promote MSCs homing in DN. Moreover, our recent study showed the DN related lncRNA-lncGm2115 was over-expressed in MSCs of DN, and effector Gm2115-miR-150 which formed by Gm2115 binding to miR-150 and Ago2 could increase the expression of the migration gene-Cxcr4. These suggested that effector lncGm2115-miR-150 may regulate MSCs homing by Cxcr4 related pathway in DN. We will focus on Gm2115-miR-150 in primary stem cells and db/db mice. Firstly, we will study the specific role of effector lncGm2115-miR-150 in DN. Secondly, we will investigate the effects of lncGm2115-miR-150 on cell migration of MSCs and the conditions of the DN mice. Finally, we will examine the role of Gm2115-miR-150 on Cxcr4/PI3K/Akt pathway in DN. Collectively this project may help to elucidate the physiopathological mechanism of DN, and provide a new insight into the prevention and treatment of DN.

间充质干细胞（MSCs）靶器官归巢率低是目前糖尿病肾病（DN）细胞治疗受限的重要原因，但机制不清。前期发现DN相关miRNAs 中（No.30800534）， 沉默miR-150促进MSCs肾脏归巢。近期RNA-seq等发现DN相关lncRNA Gm2115在DN MSCs增高，通过靶向miR-150和结合Ago2形成lncGm2115-miR-150功能性效应体，使转移因子Cxcr4表达增高。因此，我们推测“lncGm2115-miR-150效应体活化Cxcr4/PI3K/Akt信号途径”是影响DN MSCs归巢的重要原因。本课题以lncGm2115-miR-150效应体为切入点，原代DN MSCs和db/db小鼠为研究对象，研究Gm2115-miR-150具体模式；探讨其对DN MSCs归巢和病情影响；及对下游通路的调控。通过上述研究将有助于阐明MSCs归巢机制，为DN诊治提供突破点。

糖尿病肾病(diabetic nephropathy, DN)是糖尿病主要的微血管并发症之一, 也是终末期肾病的主要原因。间充质干细胞（mesenchymal stem cells, MSCs）靶器官归巢率低是目前DN细胞治疗受限的重要原因，但机制不清。近年来，研究者发现长非编码RNA（long noncoding RNAs，lncRNAs）参与DN的发生发展，然而，lncRNA在DN MSCs肾脏归巢中的作用及机制目前未见明确报道。因此，本课题拟探讨lncRNA对DN MSCs迁移归巢中的作用及机制。本课题以db/db小鼠和DN微环境（高糖、LPS 和AGEs）培养的MSCs作为DN体内外实验对象，采用转录组测序和qRT-PCR检测Gm2115的表达水平；FISH和核质qRT-PCR检测Gm2115的亚细胞定位；CPAT、ORF finde软件和western blot检测Gm2115的编码能力；划痕、Transwell迁移和迁移相关因子PAK1和MMP9表达水平等检测Gm2115对DN MSCs迁移能力的影响；荧光素酶实验和western blot检测Gm2115和miR-29b-1-5p的靶向关系；划痕实验、Transwell迁移实验、迁移相关因子PAK1和MMP9表达水平检测、组织荧光等检测Gm2115通过miR-29b-1-5p/Gli1/Akt途径影响MSCs迁移。结果发现，Gm2115是一种进化上保守的lncRNA，在DN小鼠肾组织和DN MSCs中均显著上调。此外，Gm2115的过表达或沉默可调节MSCs的细胞迁移能力。此外，Gm2115作为ceRNA靶向miR-29b-1-5p。结果还表明，在DN微环境下，Gm2115通过miR-29b-1-5p/Gli1/Akt途径调节MSCs细胞迁移能力。沉默Gm2115还可促进MSCs细胞迁移归巢肾组织，从而改善DN小鼠蛋白尿。综上，DN下lncRNA Gm2115 通过靶向抑制miR-29b-1-5p，从而调控miR-29b-1-5p靶基因Gli1的表达，影响下游Akt磷酸化，从而影响DN MSCs细胞迁移归巢肾组织，为DN细胞治疗提供实验室依据和新的突破点。