ABSTRACT
AIM: To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-ß2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). METHODS: Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-ß2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-ß2+empty plasmid group, BMP-6 overexpression group, and TGF-ß2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. RESULTS: Compared with the control group, the migration of RPE cells in the TGF-ß2 group was significantly enhanced. TGF-ß2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-ß2 and prevented TGF-ß2 from affecting EMT-related biomarkers (P<0.05). CONCLUSION: BMP-6 prevents the EMT in RPE cells induced by TGF-ß2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.
ABSTRACT
AIMS: Dysregulated long non-coding RNA (lncRNA) expression is closely related to neuroinflammation, leading to multiple neurodegenerative diseases. In this study, we investigated the function and regulation of lncRNA AK148321 in neuroinflammation using an in vitro lipopolysaccharide (LPS)-stimulated BV2 microglial cell system. METHODS: Expression of AK148321 was analyzed by qPCR. Inflammatory cytokine expression levels were determined by ELISA assay. The interaction between AK148321, microRNA (miRNA), and its target gene was validated by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell apoptosis was analyzed by Annexin V/PI staining. RESULTS: LPS treatment suppressed AK148321 expression in BV2 cells. Overexpression of AK148321 inhibited LPS-induced BV2 microglial cell activation and decreased the expression of inflammatory cytokine TNF-α and IL-1ß. AK148321 function as a competing endogenous RNA (ceRNA) by sponging microRNA-1199-5p (MiR-1199-5p). In LPS-stimulated BV2 cells, AK148321 exerted its inhibitory function via negatively modulating miR-1199-5p expression. Moreover, we identified that Heat Shock Protein Family A Member 5 (HSPA5) was a direct target of miR-1199-5p. RIP assay using the anti-Ago2 antibody further validated the relationship among AK148321, miR-1199-5p and HSPA5. The AK148321/miR-1199-5p/HSPA5 axis regulated the neuroinflammation in LPS-induced BV2 microglial cells. Microglial cell culture supernatant from LPS-stimulated, AK148321-overexpressing BV2 cells suppressed the cell apoptosis of mouse hippocampal neuronal cell HT22, while HSPA5 knockdown abrogated the suppression effect. CONCLUSION: Our findings suggest that AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cells through miR-1199-5p/HSPA5 axis.
