MiR-26b在糖尿病肾病系膜细胞肥大中异常表达的机制及功能研究

Glomerular mesangial cells (MCs) hypertrophy is one of the pathological character found at early stage of diabetic nephropathy(DN), which play an important role in the development of glomerulosclerosis. MicroRNAs (miRNAs) have been identified as negatively regulatory elements of their target genes at post-transcriptional level.The expression profile of miRNAs altered remarkably,which was associted with the onset of DN, however, until now the certain mechanism for miRNAs to involve in the pathogenesis of DN has not been fully resolved. Our preliminary data indicated that 1) miR-26b upregulated remarkably in both hypertrophic MCs exposure to high glucose and isolated glomeruli in DN mice as well. 2) The results from prediction of bioinformatics suggested that miR-26b gene located in an intron of host gene, and also glycogen synthase kinase 3β (GSK3β) was a potential candidate of target gene of miR-26b. Given that GSK3β paticipated in regulation of MCs growth,we hypothesize that the expression of miR-26b may be dependent on the activation of transcriptional factors of the host gene and play an important role in the pathogenesis of MCs hypertrophy via targeting GSK3β at early stage of DN. To test this hypothesis, in this study we firstly will investigate intracellular signaling transduction pathway leading to the upregulation of miR-26b in MCs exposure to high glucose condition，by using the techniques of siRNA, CHIP and mutant gene transfection ect.Futhermore the experiments will be performed to investigate 1)miR-26b inhibits GSK3βdirectly; 2)the influence of miR-26b and antagmir on the MCs hypertrophic phenotype via targeting GSK3beta and the underlying mechanism involved in by way of combination of clinical materials with in vivo and in vitro. The data of current study will be encourging to provide theoretical evidence for miR-26 antagomir serving as a theraputics strategy at early stage of DN.

肾小球系膜细胞（MCs）肥大是糖尿病肾病(DN)早期病理特征之一，是DN肾小球硬化的重要环节；微小RNA在转录后水平抑制靶基因表达，在DN早期肾组织内异常表达，与DN发病有关，但机制不清楚。我们前期发现1）miR-26b在高糖诱导的肥大MCs和DN早期小鼠肾小球内高表达；2）生物信息学预测miR-26b位于宿主基因的内含子上，且靶向糖原合成激酶3β（GSK3β）；文献证实GSK3β调节MCs生长。故我们推测：miR-26b高表达依赖其宿主基因转录因子激活，其靶向抑制GSK3β是MCs肥大的重要机制。为证实该推断，我们采用siRNA干扰、CHIP、突变基因转染等技术研究miR-26b上调的机制，并结合体内外实验和临床资料研究1)miR-26靶向抑制GSK3β；2)miR-26b及拮抗剂调控GSK3β对MCs肥大的影响及机制，研究结果有望为miR-26b拮抗剂成为DN早期干预措施提供实验依据。

肾小球系膜细胞（MC）肥大是糖尿病肾病（DN）早期病理特征之一，MC肥大导致细胞外基质持续分泌增多，是DN肾小球硬化的重要环节，miRNAs与DN密切相关；目前关于miRNAs参与高糖诱导MCs肥大的确切机制尚无明确定论。依托我们前期实验结果：DN小鼠miR-26b 和 miR-214显著上调，miR-196a和 miR-153显著下调；本课题进一步通过体内、外实验系统研究以上差异表达miRNAs对高糖诱导MCs肥大的调控作用及机制，通过外源性miRNAs抑制剂干预DN小鼠，探讨miRNAs对尿微量白蛋白尿的排泄率（UAE），、肾小球肥大、以及关键因子表达的影响；此外关于差异表达miRNAs对DN肾小管上皮-间充质转分化（EMT）的调控亦做了初步探讨；同时在DN患者肾活检组织中对体内、外细实验结果进行了验证。我们发现：1）差异表达miRNAs中，miR-26b通过其靶基因GSK3β，上调MCs肥大基因a-SMA、SM22表达水平，参与高糖诱导的MCs肥大；2）miR-214与其靶基因PTEN之间交叉对话，参与MCs肥大表型的发病，并上调细胞外基质蛋白IV型胶原的水平；外源性miR-214抑制剂干预2型DN db/db小鼠，在显著上调其靶基因PTEN的同时，降低UAE，缓解肾小球系膜区扩张，减轻肾小球病变；DN患者肾活检组织中miR-214表达显著上调，与MCs肥大基因a-SMA、SM22显著正相关，且与蛋白尿水平相关；靶基因PTEN显著下调，与DN动物实验结果相一致；3) 下调miR-196a通过靶向p27kip1这一机制,使细胞周期时相G1期比例增加,G2/M期下调，G1时相细胞体积增大，分泌表型发生改变，和IV型胶原上调一致，miR-196a /p27kip1调控高血糖导致的MCs肥大表型；4）DN时miR-153下调，通过释放对其靶基因Snail的抑制作用，下调上皮细胞标志蛋白E-cadherin表达，获得间质细胞表型，参与DN时肾间质纤维化的发病；以上结果提示miR-26b，miR-196a，miR-214，miR-153通过其靶基因GSK3β，p27kip1，PTEN，Snail参与了DN时MCs肥大表型以及肾小管EMT的调控。通过本课题研究，深入探讨DN时MCs肥大表型以及肾小管EMT的机制，为今后临床对DN合理干预、寻找DN早期治疗靶标提供理论依据。