Roles of NFκB-miR-29s-MMP-2 circuitry in experimental choroidal neovascularization

Journal of Neuroinflammation

Shengzhou Wu¹  Jia Qu¹  Ge Shan²  Dongsheng Yan¹  Xiaoyan Chen¹  Xiaoling Liu¹  Xianwei Wang¹  Bing Lin¹  Guibin Yin¹  Jingjing Cai¹ 

[1] State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health, China and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, 270 Xueyuan Road, Wenzhou, Zhejiang 325003, People’s Republic of China;School of Life Sciences & CAS Key Laboratory of Brain Function and Disease, University of Science and Technology of China, 443 Huangshan Road, Hefei, Anhui Province 230027, People’s Republic of China

关键词: Tumor necrosis factor alpha (TNFα);    microRNA-29 family (miR-29s);    Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB);    Matrix metallopeptidase-2 (MMP-2);    Choroidal neovascularization (CNV);   

Others  :  804257
DOI  :  10.1186/1742-2094-11-88

 received in 2013-12-09, accepted in 2014-05-01,  发布年份 2014

【 摘 要 】

Background

Previous reports have indicated that matrix metallopeptidase-2 (MMP-2) regulates angiogenic processes, which are involved in choroidal neovascularization (CNV). However, the regulation of MMP-2 in CNV has not been well-characterized. To gain more information about the regulation of MMP-2 in CNV, we analyzed the circuitry associated with MMP-2 regulation in a CNV model and in cell cultures, focusing on NFκB and the microRNA-29 family (miR-29s).

Methods

The CNV model was established by subjecting C57BL/6 mice to fundus photocoagulation with a krypton red laser. In choroidal-retinal pigment epithelial (RPE) tissues of the model, immunohistochemistry was used to evaluate the angiogenesis and MMP-2 expression; reverse-transcription quantitative PCR (RT-qPCR) was used to determine the levels of miR-29s; and western blot was used to analyze the protein levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inhibitor, IκBα, and its phosphorylated form, phospho-IκBα. At the cellular level, RT-qPCR was used to examine the levels of miR-29s following NFκB activation by tumor necrosis factor alpha (TNFα); and western blot and luciferase assay were used to determine the regulation of MMP-2 by miR-29s in a human RPE cell line (ARPE-19) and in an umbilical vein endothelial cell line (EA hy926).

Results

MMP-2 staining was increased in the choroidal neovascular membrane of laser-treated retina. Also, the NFκB pathway was induced in choroid-RPE tissue, as evidenced by a lower protein level of IκBα and a higher level of phospho-IκBα in the tissue homogenates than in those from non-treated eyes. During the period when the NFκB pathway was induced, reduced miR-29s were detected in the choroidal-RPE tissue of the laser-treated eyes. In cultured ARPE-19 cells, TNFα decreased miR-29a, b, and c, and the effects were rescued by NFκB decoy. In ARPE-19 and EA hy926, miR-29s mimics reduced the contents of secreted MMP-2 in the culture media. We also documented that miR-29s reduced MMP-2 3’-UTR-mediated luciferase transcription.

Conclusions

The results suggest that in CNV, NFκB activation inhibits miR-29s, which may contribute to angiogenesis by up-regulating the MMP-2 protein level in RPE cells. These observations may help in developing a strategy for resolving CNV by targeting miR-29s levels.

【 授权许可】

   
2014 Cai et al.; licensee BioMed Central Ltd.

【 图 表 】

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