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目的探讨微小RNA-214(miR-214)在狼疮性肾炎(LN)小鼠肾小球系膜细胞增殖中的作用及其相关机制。方法将MRL-Faslpr/JNju小鼠(简称LN小鼠,20只)和野生型小鼠(20只)处死,分离、培养其肾小球系膜细胞;将LN小鼠肾小球系膜细胞随机分为两组,分别转染miR-214 mimics(观察组)和NC mimics control(对照组)。采用实时荧光定量PCR法检测两种小鼠及两组肾小球系膜细胞miR-214相对表达量,采用MTT法检测两组转染1、2、3天的细胞增殖抑制率。采用生物信息学方法预测细胞中miR-214的靶基因,并经双荧光素酶报告基因试验验证,YKL-40是miR-214的靶基因。采用实时荧光定量PCR法和Western blotting法检测两组YKL-40 mRNA及蛋白相对表达量。取对数生长期的LN小鼠肾小球系膜细胞,分别转染si YKL-40和negative control,采用MTT法检测转染1、2、3天的细胞增殖抑制率。结果 LN小鼠和野生型小鼠肾小球系膜细胞miR-214相对表达量分别为0.35±0.09、1,两者比较P<0.01。观察组和对照组miR-214相对表达量分别为85.54±12.07、1,两组比较P<0.01。观察组转染1、2、3天的细胞增殖抑制率均高于对照组(P<0.05或<0.01)。与对照组比较,观察组YKL-40 mRNA及蛋白相对表达量均降低(P均<0.05)。与转染negative control比较,转染si YKL-40的LN小鼠肾小球系膜细胞转染1、2、3天的细胞增殖抑制率均升高(P<0.05或<0.01)。结论 LN小鼠肾小球系膜细胞中miR-214表达降低,上调miR-214表达可抑制其增殖;下调YKL-40表达可能是miR-214的作用机制。
Objective To investigate the role of miR-214 in the proliferation of glomerular mesangial cells in lupus nephritis (LN) mice and its related mechanisms. Methods MRL-Faslpr / JNju mice (LN mice, 20 for short) and wild type mice (20 mice) were sacrificed and their mesangial cells were isolated and cultured. LN mouse glomerular mesangial cells The mice were randomly divided into two groups: miR-214 mimics (observation group) and NC mimics control group (control group). Real-time fluorescence quantitative PCR was used to detect the relative expression of miR-214 in mesangial cells and mesangial cells of two groups. MTT assay was used to detect the cell proliferation inhibitory rate of transfected cells. The bioinformatics method was used to predict the target genes of miR-214 in cells, and YKL-40 was the target gene of miR-214 verified by dual luciferase reporter assay. The mRNA and protein expressions of YKL-40 in two groups were detected by real-time fluorescence quantitative PCR and Western blotting. The LN mouse mouse mesangial cells in logarithmic growth phase were transfected with si YKL-40 and negative control respectively. The inhibition rate of cell proliferation was detected by MTT assay. Results The relative expression levels of miR-214 in glomerular mesangial cells of LN mice and wild-type mice were 0.35 ± 0.09, respectively (P <0.01). The relative expression levels of miR-214 in the observation group and the control group were 85.54 ± 12.07, respectively, P <0.01. The inhibitory rate of cell proliferation in the observation group transfected for 1, 2 and 3 days were higher than that of the control group (P <0.05 or <0.01). Compared with the control group, the mRNA and protein expressions of YKL-40 in the observation group decreased (P <0.05). Compared with the negative control, the proliferation inhibition rate of LN glomerular mesangial cells transfected with si YKL-40 transfected for 1, 2 and 3 days were significantly increased (P <0.05 or <0.01). Conclusions The expression of miR-214 is decreased in glomerular mesangial cells of LN mice, and the up-regulation of miR-214 expression can inhibit its proliferation. Down-regulation of YKL-40 expression may be the mechanism of miR-214.