MiR-214调控Wnt/β-catenin信号通路在草酸钙结晶肾损伤中的作用机制研究

Calcium oxalate can induce renal tubular injury and interstitial fibrosis, which may lead to chronic kidney disease or even end-stage renal disease (ESRD). It is found that miR-214 in renal tissue is significantly upregulated in calcium oxalate crystal induced renal injury animal models. Bioinformatic analysis showed that miR-214 could regulate the expression of GSK3β and DVL1 in Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling pathway regulates the phenotype of renal tubular cells, which is closely associated with renal fibrosis. Therefore, we hypothesis that calcium oxalate crystals could injury the renal tubular cells to induce miR-214 secretion, which regulate the expression of GSK3βand DVL1 in the Wnt/β-catenin signaling pathway. To prove this hypothesis, the effect of miR-214/Wnt/β-catenin to induce tubulointerstitial injury and the detailed molecular mechanisms will be investigated by applying sequential biological methods, including flowcyte, qRT-PCR, transfection, WB and immunostaining. Not only cell lines and animal models, but also clinical specimens are used in this study. The findings of this research will broaden the clinical applications of miR-214 and guide individualized treatment of calcium oxalate renal injury.

草酸钙结晶所致的肾小管损伤、间质纤维化可导致慢性肾脏疾病甚至终末期肾病。研究发现，草酸钙结晶肾损伤动物模型中肾组织miR-214明显升高，生物信息学预测发现Wnt/β-catenin信号通路中的GSK3β、DVL1是miR-214的潜在靶点。Wnt/β-catenin信号通路调控肾小管上皮细胞表型，与肾纤维化密切相关。据此我们提出假说：草酸钙结晶损伤肾小管上皮细胞导致miR-214表达升高，通过抑制GSK3β、DVL1表达，活化Wnt/β-catenin信号通路，导致肾纤维化。为验证假说，本项目通过临床标本、细胞和动物模型，采用qPCR、细胞转染、WB、免疫荧光等技术，研究miR-124通过调控Wnt/β-catenin信号通路导致肾小管损伤、间质纤维化的发生的分子机制。预期研究结果将为阐明草酸钙结晶肾损伤的分子机制、早期诊断和治疗提供新的理论和实践依据，具有重要的临床意义。

草酸钙结晶所致的肾小管损伤、间质纤维化可导致慢性肾脏疾病甚至终末期肾病，本课题主要探索草酸钙结晶肾损伤的发病机制。前期研究通过高通量基因芯片技术发现疾病动物模型中肾组织miR-214明显升高，本课题经临床肾活检组织样本检测证实miR-214水平显著高于正常对照组患者，且与病情活动具有一定的相关性，尿miR-214水平与正常对照组患者无显著差异。通过多个microRNA靶基因数据库重新预测miR-214的潜在靶点，发现hsa-miR-214（非rno-miR-214）与GSK3B、DVL1的8mer sites和7mer sites结合位点保守性差，因此，课题组对草酸钙结晶肾损伤动物模型肾组织进行了mRNA测序（RNA-Seq）及生物信息学分析，进一步寻找疾病发展过程中关键的调控通路。此外，本课题围绕草酸钙结晶肾损伤，对临床病例进行了全外显子测序，发现了数个相关致病性不明的基因变异，拟在下一阶段作家系验证及功能试验研究，旨在揭示基因遗传背景在草酸钙结晶肾损伤中的作用及机制。