Department of Nephrology, The Second Affiliated Hospital of Guangxi Medical University, Guangxi Zhuang Autonomous Region, Nanning, China.
Department of Pathophysiology, School of Preclinical Medicine, Guangxi Zhuang Autonomous Region, Guangxi Medical University, Nanning, China.
J Biochem Mol Toxicol. 2024 Sep;38(9):e23818. doi: 10.1002/jbt.23818.
Renal fibrosis (RF) is a typical pathological presentation of end-stage chronic kidney disease (CKD) and autosomal dominant polycystic kidney disease (ADPKD). However, the precise regulatory mechanisms governing this re-expression process remain unclear. Differentially expressed microRNAs (miRNAs) associated with RF were screened by microarray analysis using the Gene Expression Omnibus (GEO) database. The miRNAs upstream of the genes in question were predicted using the miRWalk database. The miRNAs involved in the two GEO data sets were intersected to identify key miRNAs; their regulatory pathways were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Subsequently, the effects and the underlying mechanisms of target miRNA on RF were examined in a unilateral ureteral obstruction (UUO)-induced mice renal fibrotic model and a transforming growth factor-β1 (TGF-β1)-induced tubular epithelium (HK-2) fibrotic cell model. In total, 109 and 32 differentially expressed miRNAs were identified in the GSE133530 and GSE80247 data sets, respectively. GREM1 was identified as a hub gene, where its 2196 upstream miRNAs were predicted; miR-574-5p was found to be downregulated and closely related to fibrosis after data set intersection and enrichment analyses, thus was selected for further investigation. A differential expression heatmap (GSE162794) showed that miR-542-5p was downregulated. The expression of GREM1 mRNA was upregulated, whereas that of miR-542-5p was downregulated in UUO mice and fibrotic HK-2 cells as compared with the relevant controls. The binding site of miR-542-5p was predicted at the 3'UTR region of GREM1 and was confirmed by subsequent dual luciferase reporter gene assay. Western blot analysis showed that Gremlin-1 and Fibronectin were significantly upregulated after induction of TGF-β1; when miR-542-5p was overexpressed or GREM1 mRNA was interfered, the upregulations of Gremlin-1 and Fibronectin were significantly reduced. Our research demonstrates that miR-542-5p plays a critical role in the progression of RF, and thus may be a promising therapeutic target for CKD and ADPKD.
肾纤维化（RF）是终末期慢性肾脏病（CKD）和常染色体显性多囊肾病（ADPKD）的典型病理表现。然而，调控这一重新表达过程的确切机制尚不清楚。本研究通过基因表达综合数据库（GEO）数据库的微阵列分析筛选与 RF 相关的差异表达 microRNAs（miRNAs）。利用 miRWalk 数据库预测相关基因上游的 miRNAs。将两个 GEO 数据集的 miRNAs 进行交集，以确定关键 miRNAs；利用基因本体论（GO）和京都基因与基因组百科全书（KEGG）富集分析探讨其调控途径。随后，在单侧输尿管梗阻（UUO）诱导的小鼠肾纤维化模型和转化生长因子-β1（TGF-β1）诱导的肾小管上皮（HK-2）成纤维细胞模型中检测靶 miRNA 对 RF 的作用及其潜在机制。在 GSE133530 和 GSE80247 数据集中共鉴定到 109 个和 32 个差异表达的 miRNAs。GREM1 被鉴定为枢纽基因，共预测到其 2196 个上游 miRNAs；经数据集中交和富集分析发现，miR-574-5p 下调且与纤维化密切相关，因此被选为进一步研究的对象。差异表达热图（GSE162794）显示 miR-542-5p 下调。与相应对照组相比，UUO 小鼠和纤维化 HK-2 细胞中 GREM1mRNA 表达上调，而 miR-542-5p 表达下调。miR-542-5p 的结合位点在 GREM1 的 3'UTR 区被预测，并通过后续双荧光素酶报告基因实验得到验证。Western blot 分析显示，TGF-β1 诱导后 Gremlin-1 和 Fibronectin 显著上调；当过表达 miR-542-5p 或干扰 GREM1mRNA 时，Gremlin-1 和 Fibronectin 的上调显著降低。本研究表明，miR-542-5p 在 RF 进展中发挥关键作用，因此可能是 CKD 和 ADPKD 的有前途的治疗靶点。
