长链非编码RNA Erbb4-IR靶向抑制抗纤维化转录因子KLF15在慢性肾衰竭中的作用及机制研究

To inhibit renal fibrosis is essential for treating chronic renal failure. We reported previously that knockdown of lncRNA Erbb4-IR played a role in alleviation of renal fibrosis. Our preliminary data show that lncRNA Erbb4-IR could induced by AngⅡ.Knockdown of lncRNA Erbb4-IR contributed to inhibition of AngⅡinduced Collagen I expression and restoration of KLF15. A binding site of lncRNA Erbb4-IR on 5’UTR of KLF15 genomic sequence was predicted by bioinformatics analysis. Then we made a hypothesis that AngⅡinduced lncRNA Erbb4-IR promotes fibrosis by inhibition of KLF15 via targeting on its 5’UTR of genomic sequence. We planned to use in situ hybridization、nucleoplasmic separation and Real-time PCR to detect the expression of lncRNA Erbb4-IR on renal tissue and cells; to explore the role of lncRNA Erbb4-IR on AngⅡinduced pro-fibrogenesis in mouse tubular epithelial cell/ mesangial cell and 5/6 nephrectomy mouse model by siRNA and ultrasound mediated gene transfer technique; to clarify whether lncRNA Erbb4-IR interacts on 5’UTR of KLF15 genomic sequence by luciferase assay and ChIRP. We aim to explore potential therapeutic target of chronic renal fibrosis in this project.

抑制肾脏纤维化是治疗慢性肾衰竭的关键。前期发现下调lncRNA Erbb4-IR抑制肾脏纤维化。预实验发现该lncRNA可由AngⅡ上调，沉默该lncRNA后AngⅡ诱导的Collagen I受抑制及KLF15有所恢复。通过生物信息学预测KLF基因5’UTR存在该lncRNA结合位点。由此提出科学假说AngⅡ诱导的lncRNA Erbb4-IR靶向于KLF15基因5’UTR抑制其转录而促纤维化。拟用原位杂交、核浆分离、Real-time PCR观察该lncRNA在肾组织和细胞中的表达；用siRNA和超声微泡介导转基因技术沉默该lncRNA，分别在AngⅡ诱导的肾小管上皮细胞/系膜细胞促纤维化模型和5/6肾切除小鼠上观察其对肾脏纤维化和KLF15的影响并检测肾功能；通过双荧光酶报告基因分析及ChIRP明确该lncRNA是否作用于KLF15基因5’UTR。本项目旨在为延缓慢性肾衰竭提供新思路。

肾间质纤维化是多种慢性肾脏病进展至终末期的共同病理改变，延缓肾间质纤维化具重要意义。我们在多个肾间质纤维化模型中探讨非编码RNA的作用和机制。我们在小鼠5/6肾切除慢性肾衰竭模型、血管紧张素Ⅱ（Ang Ⅱ）致小鼠肾小管上皮细胞促纤维化模型和顺铂致小鼠肾间质纤维化模型中探讨长链非编码RNA（lncRNA）Erbb4-IR的表达和作用，并探讨其与抗纤维化转录因子KLF15之间的关系。我们发现5/6肾切除慢性肾衰竭模型中lncRNA Erbb4-IR表达升高；顺铂诱导的肾间质纤维化模型中lncRNA Erbb4-IR表达升高和KLF15表达下调；用AngⅡ刺激小鼠肾小管上皮细胞，lncRNA Erbb4-IR、Collagen I表达上调，KLF15表达下调；使用siRNA沉默lncRNA Erbb4-IR表达 后，AngⅡ诱导的Collagen I表达上调受抑制、KLF15表达下调有所恢复，提示lncRNA Erbb4-IR促进肾间质纤维化。我们对顺铂致间质纤维化肾脏行RNA高通量测序及PCR验证，发现顺铂组差异表达lncRNA有387个并验证了排名前两位lncRNA表达；差异表达mRNA2427个，其中补体C3在差异表达mRNA中排位第一；通过基因本体（GO）富集分析发现，与免疫相关的多个条目排位前20内；通过京都基因和基因组数据库（KEGG）富集分析发现，系统性红斑狼疮通路排位第一，其中包含了补体级联反应通路；通过实时定量PCR验证发现顺铂组补体C1q、C2、C3和C4 mRNA表达均升高，提示顺铂诱导的肾间质纤维化中存在补体活化。通过单侧输尿管结扎模型及TGF-β1诱导肾小管上皮细胞促纤维化模型，探讨miR-140-5p在纤维化中的作用，发现miR-140-5p抑制TGF-β1诱导肾小管上皮细胞促纤维化反应。以上研究结果多角度为肾间质纤维化防治提供了新靶点。