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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。
Objective To investigate whether miR-155 affects the expression of matrix metalloproteinase 2 (MMP-2) and MMP-9 in renal tubular epithelial cells by downregulating the target gene Kruppel-like factor 4 (KLF4). Methods Firstly, we verified whether miR-155 could inhibit the expression and activity of MMPs in renal tubular epithelial cells and its effect on KLF4 expression: miR-155-mimic, miR-155mimic-control (empty plasmid), blank medium transfected in vitro The expression of MMP-2, MMP-9 and KLF4 were detected by Western blot. Gelatin zymography was used to detect the expression of MMP-2 And MMP-9 activity. Bioinformatics predicts miR-155 potential target gene after KLF4, to verify whether miR-155 through targeted inhibition of KLF4 play its role: tubular epithelial cells with miR-155 overexpression plasmid transfected, and then further transfected with KLF4 Overexpression of plasmid or empty plasmid, 6h after Western blot and gelatin zymography again test the above indicators. The other cells were used to construct luciferase plasmids containing wild-type and mutant KLF4-3’-UTR sequences, respectively, co-transfected with miR-155-mimic or empty plasmid control into renal tubular epithelial cells. Luciferase assay Targeted inhibition of KLF4 gene by miR-155. Results miR-155 was up-regulated in miR-155-mimic-transfected cells compared with miR-155mimic-transfected cells. The expression of KLF4 was inhibited and the expression and activity of MMP-2 and MMP-9 were decreased. Compared with transfected miR-155 plasmid and transfected miR-155 + empty plasmid, the expression of KLF4 in transfected cells over-expressing KLF4 + miR-155 plasmid increased the expression and activity of MMP-2 and MMP-9. Luciferase Assay Validated miR-155 Targeted to KLF4. Conclusions MiR-155 can down-regulate KLF4 through targeting and inhibit the expression and activity of MMP-2 and MMP-9 in renal tubular epithelial cells.