Figure 5.

The functional role of tRF-Glu-CTC is associated with angiogenin (ANG)-involved tsRNA generation and the secretion of inflammatory factors. (A) The ANG expression was upregulated under hypoxia condition (n = 3). (B) The ANG expression was upregulated in RPE-choroid complex after CNV formation (n = 5). (C) The expression level of tRF-Glu-CTC decreased after the suppression of ANG in hypoxia condition, whereas the level revealed no difference in normoxia condition (n = 3, one-way ANOVA test). (D) Immunofluorescence staining revealed that ANG was colocalized with IB4+ neovascular region. Scale bar, 50µm. (E) The Gene Ontology (GO) functional enrichment analysis demonstrated the top 20 biological process (BP) terms enriched in the differentially expressed genes (DEGs). (F) Schematic diagram of the overlap between the top 50 enriched BPs of DEGs and enriched BPs of predicted target genes of tRF-Glu-CTC. (G) The volcano plot of DEGs based on a threshold of fold change > 1.5 or < 0.67 and P < 10^-5. (H & I) The expression levels of related inflammatory factors significantly increased in HUVECs transfected with tRF-Glu-CTC mimic (n = 3) and RPE-choroid complex 4 d after CNV formation (n = 5). (J & M) The choroidal sprouting assay revealed that sprouting area was elevated by culture medium collected from tRF-Glu-CTC transfected HUVECs (n = 6). Scale bar, 500 µm. (K & N) The tube formation assay revealed that the total length and total branches length of HUVECs were reduced after 24 h culture with medium from tRF-Glu-CTC mimic transfected HUVECs (n = 3). Scale bar, 250 µm. (L & O) The transwell assay revealed that the migrated cells significantly increased when HUVECs transfected with tRF-Glu-CTC mimic were added to the bottom chamber (n = 4). Scale bar, 250 µm. *P < 0.05, **P < 0.01, ***P < 0.001. All data were based on at least three independent experiments and were demonstrated as means ± SEM.