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、AngⅡ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AngⅡ组和空白组细胞中miR-381表达变化,miR-381组、NC组及AngⅡ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);AngⅡ刺激NRK49F细胞后,相比空白组miR-381相对表达量降低,而AngⅡ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。
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,AngⅡgroup 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.AngⅡwas then used to stimulate the cells in the first three groups,and the expression changes of miR-381 in the AngⅡgroup and the blank control group were detected,and we also investigated the alteration ofα-SMA expression in the miR-381 transfection group,NC group and AngⅡgroup after the stimulation of AngⅡ.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 factorsα-SMA,CTGF,COL1A1 and COL3A1 in the miR-381 transfection group were reduced to different degrees(P<0.05).After the stimulation of AngⅡon NRK49F cells,the expression of miR-381 was significantly lower than that of the blank control group,while the expression level ofα-SMA increased in the AngⅡgroup 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.
作者
王立成
李现铎
陈冬冬
唐冠宝
门同义
Li-cheng Wang;Xian-duo Li;Dong-dong Chen;Guan-bao Tang;Tong-yi Men(Department of Urology,Shandong Provincial Qianfoshan Affilaiated Hospital to Shandong University,Jinan,Shandong 250014,China)
出处
《中国现代医学杂志》
CAS
2020年第16期1-6,共6页
China Journal of Modern Medicine
