Pharmacology Department, Cangzhou central hospital, Cangzhou, Hebei, China.
Oncology Surgery, Cangzhou central hospital, Cangzhou, Hebei, China.
Sci Rep. 2024 Oct 30;14(1):26083. doi: 10.1038/s41598-024-77691-1.
To investigate the role and molecular mechanism of miR-126 in unilateral ureteral occlusion (UUO). We used bioinformatics to analyse miRNAs specifically expressed in UUO. The mouse model of UUO was established using RAW264.7 cells cultured in vitro and in vivo. The mice were divided into control group, miR-126-NC (negative control) group and miR-126-KD (knockdown) group. Then the relative expression of miR-126 was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the renal fibrosis was detected by Masson staining, and the protein expression of CD68, collagen I and collagen III in the kidney was detected by immunofluorescence assay. Immunohistochemistry detects α-SMA expression. Moreover, Western blotting was performed to measure the expressions of p-PI3K, CD163, CD206, CD86, iNOS, IL-1β, p-FAK, p-Rac-1, p-IRS-1 and MMP9. The relative fluorescence intensity of F-actin and p-FAK was detected by immunofluorescence assay, and the phagocytosis ability of macrophages was determined by phagocytosis assay with fluorescent microspheres. Bioinformatics analysis reveals miR-126-specific overexpression in UUO. Successful transfection of miR-126-NC and miR-126-KD was confirmed by RT-PCR. The selective reduction of miR-126 was validated by Masson, immunohistochemistry and immunofluorescence staining to decrease the area of UUO-induced renal fibrosis and to lower the expression of CD68, α-SMA, collagen I, and collagen III. The reduction of iNOS expression may also be achieved with selective knockdown of miR-126, as verified by cell tests. enhances the phagocytic ability of macrophages and the expression of p-PI3K, CD206, p-FAK, F-actin, p-Rac-1, p-IRS-1 and MMP9. MiR-126 can inhibit the PI3K signaling pathway, promote M1 macrophage polarization, and suppress the activation of FAK and Rac-1, thus accelerating the progression of UUO.
研究 miR-126 在单侧输尿管梗阻 (UUO) 中的作用及分子机制。方法：利用生物信息学分析 UUO 中特异性表达的 miRNA。体外培养 RAW264.7 细胞构建 UUO 小鼠模型，并进行体内实验。将小鼠分为对照组、miR-126-NC（阴性对照）组和 miR-126-KD（敲低）组。采用实时荧光定量聚合酶链反应 (qRT-PCR) 检测 miR-126 的相对表达量，Masson 染色检测肾纤维化程度，免疫荧光法检测肾脏 CD68、Ⅰ型胶原和Ⅲ型胶原的蛋白表达。免疫组化检测α-SMA 表达。Western blot 检测 p-PI3K、CD163、CD206、CD86、iNOS、IL-1β、p-FAK、p-Rac-1、p-IRS-1 和 MMP9 的表达。免疫荧光法检测 F-actin 和 p-FAK 的相对荧光强度，通过吞噬荧光微球实验检测巨噬细胞的吞噬能力。结果：生物信息学分析显示 miR-126 在 UUO 中特异性过表达。通过 RT-PCR 证实 miR-126-NC 和 miR-126-KD 转染成功。Masson、免疫组化和免疫荧光染色证实 miR-126 的选择性敲低可减少 UUO 诱导的肾纤维化面积，降低 CD68、α-SMA、Ⅰ型胶原和Ⅲ型胶原的表达。选择性敲低 miR-126 还可降低 iNOS 表达，细胞实验证实。增强巨噬细胞的吞噬能力和 p-PI3K、CD206、p-FAK、F-actin、p-Rac-1、p-IRS-1 和 MMP9 的表达。结论：miR-126 可抑制 PI3K 信号通路，促进 M1 型巨噬细胞极化，抑制 FAK 和 Rac-1 的激活，从而加速 UUO 的进展。
