Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, 28 Fuxing Road, Beijing, China.
Stem Cell Res Ther. 2021 Mar 10;12(1):171. doi: 10.1186/s13287-021-02210-2.
Renal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.
We performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.
miR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70-100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.
miR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.
肾间质纤维化是肾脏衰老和慢性肾衰竭的一个重要病理特征，它由间充质干细胞（MSCs）调控。我们之前已经证明，在老年大鼠的 MSC 来源的细胞外囊泡（MSC-EVs）中，miR-133b 的表达水平较低。然而，miR-133b 可以介导转化生长因子-β1（TGF-β1）诱导的肾小管上皮-间质转化（EMT）的抑制。我们研究了 miR-133b 治疗老年肾间质纤维化的效果，并评估了其靶基因。
我们通过实时聚合酶链反应检测不同浓度（0、6、8 和 10ng/ml）和不同时间点（0、24、48 和 72h）TGF-β1 诱导的 HK2 细胞 EMT 过程中 miR-133b 的表达。使用双荧光素酶报告基因实验验证 miR-133b 的靶基因。在体外实验中，我们通过转染 miR-133b 后刺激 HK2 细胞，检测 miR-133b 靶基因 E-钙黏蛋白、α-平滑肌肌动蛋白（SMA）、纤连蛋白和胶原 3A1（Col3A1）的 mRNA 和蛋白表达。建立了 24 月龄单侧输尿管梗阻（UUO）小鼠模型，并将转染试剂和 miR-133b 注入尾静脉。在第 7 和 14 天检测 miR-133b 的靶基因以及其他参数，如 mRNA 和蛋白表达水平以及肾间质纤维化。
miR-133b 的表达随着 TGF-β1 浓度和处理时间的增加而逐渐降低，miR-133b 模拟物下调结缔组织生长因子（CTGF）的表达。双荧光素酶报告基因实验证实 CTGF 是 miR-133b 的直接靶基因。转染 miR-133b 模拟物抑制了 TGF-β1 诱导的 HK2 细胞 EMT；这种作用被 CTGF 的过表达所逆转。miRNA-133b 表达在注射 miRNA-133b 过表达复合物后在小鼠肾脏组织中显著增加（约 70-100 倍），显著减轻了 UUO 老年小鼠的肾间质纤维化。
miR-133b 对 CTGF 表达有靶向抑制作用，从而减少 TGF-β1 诱导的 HK2 细胞 EMT 和 UUO 老年小鼠的肾间质纤维化。
