MiR-155与 KLF4的靶向结合对肾小管上皮细胞MMPs表达的影响

【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 -155 mimic-对照（空质粒）、空白培养基转染离体培养的肾小管上皮细胞，6 h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，6 h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， 24 h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 -155 mimic-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。

MiR-155 Inhibits the Expression of MMPs in HK2 Cells by Targeting KLF4

【Abstract】 Objective To determine whether miR -155 inhibits the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) in HK2 cells by targeting Kruppel-like factor 4 (KLF4). Methods MiR -155-mimic, miR -155-NC empty plasmid, and culture medium were transfected into renal tubular epithelial cells, respectively. Six hours later, the expression of miR -155 was detected by real time-PCR; the expressions of MMP-2, MMP-9 and KLF4 were detected by Western blot; and the activity of MMP-2 and MMP-9 in the cells was detected by gelatin zymography. Because KLF4 was predicted as the target gene of miR -155 by bioinformatics. The miR-155 overexpressed HK2 cells were transfected with KLF4 overexpression plasmid or empty plasmid. Six hours later, the expressions of MMP-2, MMP-9 and KLF4, and the activity of MMP-2 and MMP-9 were measured again. Finally, cells containing luciferase plasmids with KLF4-3′-UTR wild type (WT) or mutant (MUT) sequence were constructed and transfected with miR -155-mimic or empty plasmid. Luciferase assay was used to confirm whether KLF4 -3′-UTR was the binding site in targeting miR -155．Results Compared with the cells transfected with empty plasmid, the expression of miR -155 was up-regulated and the expressions of MMP-2, MMP-9 and KLF4 were down-regulated in the cells transfected with miR -155-mimic. Compared with the cells transfected with miR -155 mimic or miR -155 mimic+empty plasmid, the expressions of MMP-2, MMP-9 and KLF4 were up-regulated in the KLF4+miR -155 transfected cells. Luciferase assays confirmed that miR -155 binds to KLF4 , and KLF4 -3′-UTR is the target gene of miR -155. Conclusion MiR-155 inhibits the expressions of MMP-2 and MMP-9 in HK2 cells by targeting KLF4 -3′-UTR.