Department of General Practice, The First Affiliated Hospital of Hainan Medical University, Hainan 570102, China.
Hainan Medical University, Hainan 570000, China.
Dis Markers. 2023 Feb 16;2023:4397829. doi: 10.1155/2023/4397829. eCollection 2023.
This study investigated the mechanism of microRNA (miRNA, miR) in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) involved in renal function in vivo and in vitro injury repair of rat primary kidney cells (PRKs).
Gene Expression Omnibus analysis of potential target miRNAs in nephrotic rats. Real-time quantitative polymerase chain reaction verified the correlation of these miRNAs and screened the effective target miRNAs and their downstream putative target mRNAs. Western blot analyzes the protein levels of DEAD-box helicase 5 (DDX5) and the activation of the proapoptotic factor caspase-3/9 (cleaved). Dil-Ac-LDL staining, immunofluorescence, and a transmission electron microscope (TEM) were used to identify the successful isolation of EPCs and PRKs and the morphology of MVs. Cell Counting Kit-8 was used to detect the effect of miRNA-mRNA on the proliferation of PRKs. Standard biochemical kits were used to detect biochemical indicators in rat blood and urine. Dual-luciferase analysis of miRNA binding to mRNA was conducted. The effect of miRNA-mRNA interaction on the apoptosis level of PRKs was analyzed by flow cytometry.
A total of 13 rat-derived miRNAs were potential therapeutic targets, and miR-205 and miR-206 were screened as the targets of this study. We found that the EPC-MVs alleviated the increase of blood urea nitrogen and urinary albumin excretion and the decrease in creatinine clearance caused by hypertensive nephropathy in vivo. The effect of MVs in improving renal function indicators was promoted by miR-205 and miR-206 and inhibited by knockdown of expressed miR-205 and miR-206. In vitro, angiotensin II (Ang II) promoted growth inhibition and apoptosis of PRKs, and similarly, dysregulated miR-205 and miR-206 affected the induction of Ang II. We then observed that miR-205 and miR-206 cotargeted the downstream target DDX5 and regulated its transcriptional activity and translational levels, while also reducing the activation of proapoptotic factors caspase-3/9. Overexpressed DDX5 reversed the effects of miR-205 and miR-206.
By upregulating the expression of miR-205 and miR-206 in MVs secreted by EPC, the transcriptional activity of DDX5 and the activation of caspase-3/9 can be inhibited, thereby promoting the growth of PRKs and protecting the injury caused by hypertensive nephropathy.
本研究探讨了内皮祖细胞（EPC）分泌的微小 RNA（miRNA，miR）在体内和体外损伤修复大鼠原代肾细胞（PRK）中参与肾功能的机制。
进行了肾病大鼠中潜在靶 miRNAs 的基因表达组学分析。实时定量聚合酶链反应验证了这些 miRNAs 的相关性，并筛选了有效的靶 miRNAs 及其下游假定靶 mRNAs。Western blot 分析 DEAD -box 解旋酶 5（DDX5）的蛋白水平和促凋亡因子 caspase-3/9（裂解）的激活。Dil-Ac-LDL 染色、免疫荧光和透射电镜（TEM）用于鉴定 EPC 和 PRK 的成功分离和 MV 的形态。细胞计数试剂盒-8 用于检测 miRNA-mRNA 对 PRK 增殖的影响。标准生化试剂盒用于检测大鼠血液和尿液中的生化指标。进行 miRNA 与 mRNA 结合的双荧光素酶分析。通过流式细胞术分析 miRNA-mRNA 相互作用对 PRK 凋亡水平的影响。
共鉴定出 13 种大鼠来源的 miRNAs 作为潜在的治疗靶点，筛选出 miR-205 和 miR-206 作为本研究的靶点。我们发现，EPC-MVs 可减轻体内高血压肾病引起的血尿素氮升高、尿白蛋白排泄增加和肌酐清除率降低。miR-205 和 miR-206 促进 MV 改善肾功能指标的作用，下调表达的 miR-205 和 miR-206 则抑制该作用。在体外，血管紧张素 II（Ang II）促进 PRK 的生长抑制和凋亡，同样，失调的 miR-205 和 miR-206 也影响 Ang II 的诱导。我们观察到，miR-205 和 miR-206 共同靶向下游靶标 DDX5，并调节其转录活性和翻译水平，同时降低促凋亡因子 caspase-3/9 的激活。过表达 DDX5 逆转了 miR-205 和 miR-206 的作用。
通过上调 EPC 分泌的 MV 中 miR-205 和 miR-206 的表达，可以抑制 DDX5 的转录活性和 caspase-3/9 的激活，从而促进 PRK 的生长，保护高血压肾病引起的损伤。
