MiR-155与KLF4的靶向结合对肾小管上皮细胞MMPs表达的影响

发布时间：2018-04-30 19:06

本文选题：慢性肾功能不全 + 微小RNA　；参考：《四川大学学报(医学版)》2017年02期

【摘要】：目的研究miR-155是否通过下调靶基因Kruppel样因子4(KLF4)影响肾小管上皮细胞中基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)和MMP-9的表达。方法首先验证miR-155是否可以抑制肾小管上皮细胞中MMPs的表达和活性及其对KLF4表达影响:miR-155-mimic、miR-155mimic-对照(空质粒)、空白培养基转染离体培养的肾小管上皮细胞,6h后,荧光定量RT-PCR检测miR-155表达,免疫印迹试验(Western blot)检测细胞MMP-2、MMP-9、KLF4的表达,明胶酶谱检测细胞中MMP-2和MMP-9的活性。生物信息学预测miR-155潜在靶基因为KLF4后,验证miR-155是否是通过靶向抑制KLF4发挥其作用:肾小管上皮细胞用过表达miR-155的质粒转染后,再进一步转染KLF4过表达质粒或空质粒,6h后Western blot和明胶酶谱法再次检测上述指标。另取细胞,分别构建包含KLF4-3′-UTR野生型和突变型序列的荧光素酶质粒,与miR-155-mimic或者空质粒对照共转染肾小管上皮细胞,24h后荧光素酶试验检测miR-155对KLF4基因的靶向抑制。结果相比转染miR-155mimic-对照的细胞,转染了miR-155-mimic的细胞miR-155表达上调,KLF4的表达受到抑制,MMP-2、MMP-9的表达和活性下降。相比于转染miR-155质粒和转染miR-155+空质粒,转染过表达KLF4+miR-155质粒的细胞KLF4表达上调,MMP-2、MMP-9的表达和活性增加。荧光素酶试验验证miR-155可与KLF4靶向结合。结论 MiR-155可以通过靶向作用下调KLF4,从而抑制肾小管上皮细胞中MMP-2、MMP-9的表达和活性。
[Abstract]:Objective to investigate the effect of miR-155 on the expression of matrix metalloproteinase (MMP) 2(matrix metalloproteinase _ 2 (MMP-2) and MMP-9 in renal tubular epithelial cells by down-regulation of target gene Kruppel like factor 4 (KLF4). Methods to investigate whether miR-155 could inhibit the expression and activity of MMPs in renal tubular epithelial cells and its effect on the expression of KLF4. The expression of miR-155 was detected by fluorescence quantitative RT-PCR after transfection with empty plasmid and blank medium for 6 hours. The expression of MMP-2, MMP-9 and KLF4 was detected by Western blotting, and the activity of MMP-2 and MMP-9 was detected by gelatin zymogram. Bioinformatics predicts the potential target of miR-155 because of KLF4, and verifies whether miR-155 plays its role by targeting the inhibition of KLF4: renal tubular epithelial cells are transfected with plasmids expressing miR-155. Western blot and gelatin zymogram were used to detect the above indexes again after further transfection of KLF4 overexpression plasmids or empty plasmids for 6 h. Luciferase plasmids containing wild-type and mutants of KLF4-3'-UTR were constructed and cotransfected with miR-155-mimic or empty plasmid for 24 hours. The targeting inhibition of KLF4 gene by miR-155 was detected by luciferase assay. Results compared with those transfected with miR-155 mimic-control cells, the expression of miR-155 in miR-155-mimic transfected cells was inhibited and the expression and activity of MMP-2 / MMP 9 decreased. Compared with the transfection of miR-155 plasmid and empty miR-155 plasmid, the expression and activity of MMP-2MMP-9 were up-regulated in the cells transfected with KLF4 miR-155 plasmid. Luciferase assay confirmed that miR-155 could bind to KLF4. Conclusion MiR-155 can down-regulate the expression and activity of MMP-2 and MMP-9 in renal tubular epithelial cells by targeting KLF4.
【作者单位】：兰州大学第二医院肾内科;兰州大学第二医院风湿科;
【基金】：甘肃省自然科学基金项目(No.1308RJZA246) 甘肃省卫生行业科研计划项目(No.GSWST2013-02)资助
【分类号】：R692

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