Purpose
To prove whether bone marrow-derived cells(BMCs), which are involved in the development of choroidal neovascularization (CNV) , are the main source of matrix metalloproteinases (MMPs) , and whether the MMP-2/-13 expression is potentially regulated by miR188-5p
Methods
BMCs of GFP transgenic mice were transplanted to wild-type C57 mice to establish chimeras, and CNV was induced by laser. MMP-2/MMP-13 levels in hypoxia-treated bone marrow-derived mesenchymal stem cells (BMSCs) and in CNV eyes were quantified by western blot. miR-188-5p levels were detected using qRT-PCR. Immunofluorescence staining (IF) was used to identify MMP-2/MMP-13 and miR188-5p expressed by GFP -positive BMCs or cells in situ in CNV, and the expression levels were semi-quantified by images analysis.
Results
In vitro, expression of miR-188-5p decreased, while the mRNA and protein levels of MMP-2/13 significantly increased in hypoxia-treated BMSCs (p < 0.05). In vivo, western blot and IF showed MMP-2 expression rapidly increased after CNV induction and reached the climax on day 3, when the corresponding expression in BMCs reached the peak (64.21% of the total expression in CNV). MMP-13 expression showed relative slow increase at the early stage of CNV and reached the climax on day 7, meanwhile the expression in BMCs also reached the highest 79.61% of the total expression. qRT-PCR and IF results demonstrated that miR188-5p expression fell rapidly after laser, and the lowest expression level was observed on day 7. MMP-2 / MMP-13 protein expression in CNV was negatively correlated with miR188-5p expression. Similarly, IF semi-quantitative analysis of the expression of MMP-13 in BMCs in CNV showed a negative correlation with the expression of miR188-5p in BMCs (r=-0.868,p<0.05). While the expression of MMP-2 in BMCs showed a negative correlation with miR188-5p only in the first 5 days after CNV induction (r=-0.997,p<0.05)(Figure). The predict result of target genes showed that 3’UTRs of MMP-2 and MMP-13 have complementary association points of miR188-5p.
Conclusions
In experimental CNV, MMPs partially expressed by BMCs. BMCs may play the main role of regulating MMPs expression in CNV, and the expression of MMP-2/MMP-13 from BMCs might be regulated by miR188-5p. Further investigation is needed to confirm the regulation of MMPs in BMCs by miRNA.