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[MiR-155 Inhibits the Expression of MMPs in HK2 Cells by Targeting KLF4]
Ya-Xian Ning1, Jian-Qin Wang1, Xiao-Yuan Wang2
1Department of Nephrology, the Second Hospital of Lanzhou University, Lanzhou 730030, China.
Objectives:
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!d-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!d-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!d-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!d-UTR.
Insights
MicroRNA-155 (miR-155) inhibits matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) expression in HK2 cells. This inhibition occurs by targeting Kruppel-like factor 4 (KLF4) in renal tubular epithelial cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Matrix metalloproteinases (MMPs), including MMP-2 and MMP-9, play crucial roles in extracellular matrix remodeling.
- Dysregulation of MMPs is implicated in various kidney diseases.
- MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and are involved in cellular processes.
Purpose of the Study:
- To investigate the inhibitory effect of microRNA-155 (miR-155) on the expression of MMP-2 and MMP-9 in HK2 cells.
- To determine if Kruppel-like factor 4 (KLF4) is a direct target of miR-155 in this cellular context.
- To elucidate the molecular mechanism by which miR-155 regulates MMP expression.
Main Methods:
- Transfection of HK2 cells with miR-155 mimic or control plasmids.
- Quantitative real-time PCR (qRT-PCR) to measure miR-155 expression.
- Western blot analysis to assess protein levels of MMP-2, MMP-9, and KLF4.
- Gelatin zymography to evaluate MMP-2 and MMP-9 enzymatic activity.
- Bioinformatic prediction of KLF4 as a miR-155 target.
- Luciferase reporter assays to confirm direct binding of miR-155 to the KLF4 3'-untranslated region (3'-UTR).
Main Results:
- Overexpression of miR-155 led to a significant decrease in the expression of MMP-2, MMP-9, and KLF4 in HK2 cells.
- Restoration of KLF4 expression reversed the inhibitory effects of miR-155 on MMP-2 and MMP-9.
- Luciferase assays confirmed that miR-155 directly binds to the 3'-UTR of KLF4, validating KLF4 as a target gene.
Conclusions:
- MiR-155 exerts an inhibitory effect on the expression of MMP-2 and MMP-9 in HK2 cells.
- This inhibition is mediated through the direct targeting of Kruppel-like factor 4 (KLF4) by miR-155.
- The findings highlight a novel regulatory pathway involving miR-155 and KLF4 in controlling MMP expression in renal tubular epithelial cells.
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