MicroRNA-381 靶向调控KLF-4 抗肾纤维化的机制研究

MiR-381 regulates the development of renal fibrosis via KLF-4

摘要:

目的 探究MicroRNA-381（miR-381）通过靶向调控KLF-4 抑制肾纤维化病理进程的作用机制。 方法 选取2014 年2 月—2017 年12 月山东大学附属千佛山医院收治的106 例肾纤维化患者；另取同期该院 健康体检者100 例作为对照组。采用microRNA（miRNA）靶基因数据库筛选出miR-381 潜在靶基因KLF-4 并使用荧光素酶进行验证；实时荧光定量聚合酶链反应（qRT-PCR）检测肾纤维化患者血清miR-381。将 NRK49F 细胞分为miR-381 组、NC 组、Ang Ⅱ组及空白组。使用qRT-PCR 和Western blotting 检测miR-381 组、 NC 组中KLF-4 表达变化；使用qRT-PCR 检测miR-381 组和NC 组中多种肾纤维化因子mRNA 表达变化。 检测Ang Ⅱ组和空白组细胞中miR-381 表达变化，miR-381 组、NC 组及Ang Ⅱ组细胞中α-SMA 表达变化。 将小鼠随机分为单侧输卵管梗阻（UUO）组、UUO+miR-381 组及假手术组，检测UUO 组和假手术组小鼠 肾脏组织miR-381 表达变化，并使用Masson 染色观察miR-381 对肾纤维化的作用。结果 qRT-PCR 结果 显示，肾纤维化患者血清miR-381 相对表达量低于对照组（P <0.05）。相比NC 组，miR-381 组肾纤维化因子 α-SMA、CTGF、COL1A1 及COL3A1 mRNA 相对表达量降低（P <0.05）；Ang Ⅱ刺激NRK49F 细胞后，相 比空白组miR-381 相对表达量降低，而Ang Ⅱ组α-SMA 相对表达量升高，过表达miR-381 后α-SMA 相对 表达量降低（P <0.05）。KLF-4 是miR-381 靶基因，miR-381 可以负调控KLF-4 mRNA 和蛋白的表达（P <0.05）。 UUO 组小鼠肾脏组织中miR-381 相对表达量低于假手术组，相比UUO 组，UUO+miR-381 组肾纤维化区域 减小，细胞外基质沉积变少，COL1A1 和COL3A1 mRNA 相对表达量降低（P <0.05）。结论 miR-381 通过抑 制KLF-4 抗肾纤维化的发展进程，可能成为肾纤维化诊断及治疗的新靶点。

Abstract:

Objective To study the inhibitory effect of miR-381 on the pathological process of renal fibrosis by targeting KLF-4. Methods The potential target gene KLF-4 of miR-381 was screened out from the microRNA target gene database and verified with luciferase. The expression level of miR-381 in serum of patients with renal fibrosis and healthy controls was respectively detected by qRT-PCR. NRK49F cells were divided into miR-381 transfection group, NC group, Ang Ⅱ group and blank control group. The expression of KLF-4 in miR-381 transfection group and NC group was determined by qRT-PCR and Western blotting, and qRT-PCR was used to explore the mRNA expression of multiple renal fibrogenic factors in miR-381 transfection group and NC group. Ang Ⅱ was then used to stimulate the cells in the first three groups, and the expression changes of miR-381 in the Ang Ⅱ group and the blank control group were detected, and we also investigated the alteration of α-SMA expression in the miR-381 transfection group, NC group and Ang Ⅱ group after the stimulation of Ang Ⅱ . Mice were randomly divided into UUO + miR-381 group, UUO group and sham group. The expression of miR-381 in the renal tissues of the UUO group and sham group was detected and the effects of miR-381 on renal fibrosis were observed morphologically by Masson staining. Results The results of qRT-PCR showed that the expression level of miR-381 in the serum of patients with renal fibrosis was lower than that in the healthy control group (P < 0.05). Compared with the NC group, mRNA expressions of renal fibrogenic factors α-SMA, CTGF, COL1A1 and COL3A1 in the miR-381 transfection group were reduced to different degrees (P < 0.05). After the stimulation of Ang Ⅱ on NRK49F cells, the expression of miR-381 was significantly lower than that of the blank control group, while the expression level of α-SMA increased in the Ang Ⅱ group but decreased after overexpression of miR-381 (P < 0.05). KLF-4 was the target gene of miR-381, and miR-381 could negatively regulate the expression of KLF-4 mRNA and protein (P < 0.05). The renal expression level of miR-381 in UUO group was lower than that in the sham group. Compared with the UUO group, the renal fibrosis area and extracellular matrix deposition in the UUO + miR-381 group were declined, and the expression levels of COL1A1 and COL3A1 mRNA were decreased. Conclusions MiR-381 may be a new target for the diagnosis and treatment of renal fibrosis by inhibiting the development of renal fibrosis via KLF-4.