miR-29c-5p knockdown reduces inflammation and blood-brain barrier disruption by upregulating LRP6

Abstract

Blood-brain barrier participates in the pathological process of ischemic stroke. MicroRNA-29c-5p was highly expressed in clinical samples from patients with ischemic stroke. In this study, oxygen-glucose deprivation (OGD) treatment of astrocytes enhanced the permeability of brain microvascular endothelial cells (BMECs), and the miR-29c-5p expression was elevated in clinical samples from patients with ischemic stroke. For the function of miR-29c-5p in ischemic stroke, the miR-29c-5p knockdown decreased the permeability and the tight junction protein (TJP) destruction of BMECs and ameliorated the inflammation induced by OGD-treated astrocytes. Mechanistically, miR-29c-5p interacted with lipoprotein receptor-related protein 6 (LRP6) and negatively regulated the LRP6 expression in astrocytes. Moreover, the rescue assays indicated that the interference with miR-29c-5p ameliorated the TJP destruction of BMECs and inflammation caused by OGD-treated astrocytes by increasing the LRP6 expression. Together, miR-29c-5p knockdown decreased the high permeability and the TJP destruction of BMECs and ameliorated the inflammation induced by OGD-treated astrocytes by elevating LRP6 expression.

Keywords: astrocytes; blood–brain barrier; brain microvascular endothelial cells; ischemic stroke; miRNA.

Figure 1

Influence of the OGD treatment of astrocytes on the permeability of BMECs. The primary astrocytes were isolated, and the primary astrocytes were treated with OGD for 3 h and then cocultured with primary brain microvascular epithelial cells (BMECs) for 24 h. (a) An immunofluorescence staining assay was performed to verify the isolated primary astrocytes (scale bar: 100 µm). (b) The TEER value of BMECs was determined using the Millicell-ERS instrument (Millipore, MA, USA). (c) Analysis of the penetration rate of NaF. OGD: oxygen-glucose deprivation, TEER: transepithelial electrical resistance, and NaF: sodium fluorescein. Data are presented as an average of three independent assays. **P < 0.01, ***P < 0.001 vs control.

Figure 2

Effect of miR-29c-5p on the high permeability of BMECs caused by OGD-treated astrocytes. Astrocytes transfected with miR-29c-5p inhibitor or NC-inhibitor were treated with OGD for 3 h and then cocultured with BMECs for 24 h. (a) Western blot was performed to analyze the protein levels of astrocyte activation markers GFAP and GLP-1R. (b) Analysis of the TEER value of BMECs. (c) Detection of the ability of NaF to penetrate BMECs. (d) LDH release assay was performed to assess the injury of BMECs. NC: negative control, GLP-1R: glucagon-like peptide-1 receptor, and LDH: lactate dehydrogenase. Data are presented as an average of three independent assays. *P < 0.05, ***P < 0.001 vs NC-inhibitor.

Figure 3

Influence of miR-29c-5p on the TJP destruction of BMECs caused by OGD-treated astrocytes. Astrocytes transfected with miR-29c-5p inhibitor or NC-inhibitor were treated with OGD for 3 h, and the cells were cocultured with BMECs for 24 h. (a) The protein levels of TJPs claudin-5, occludin, and ZO-1 were determined using Western blot. (b) Immunofluorescence staining assay was applied to quantify the claudin-5, occludin, and ZO-1 expressions (scale bar: 100 µm). Data are presented as an average of three independent assays. **P < 0.01, ***P < 0.001 vs NC-inhibitor.

Figure 4

Effect of miR-29c-5p on the inflammation caused by OGD-treated astrocytes. Astrocytes transfected with miR-29c-5p inhibitor or NC-inhibitor were treated with OGD for 3 h. (a) ELISA was performed to test the concentrations of inflammatory cytokines IL-1β, IL-6, TNF-α, and TGF-β in cell culture supernatant of astrocytes. (b) The protein levels of inflammatory factors VEGF-A and MMP-9 were tested using Western blot. (c) An immunofluorescence assay was performed to assess the expressions of VEGF-A and MMP-9 (scale bar: 100 µm). Data are presented as an average of three independent assays. **P < 0.01, ***P < 0.001 vs NC-inhibitor.

Figure 5

Verification of the interaction between miR-29c-5p and LRP6: (a and b) Bioinformatic databases, TargetScan and Metascape, were applied to predict the possible downstream regulatory targets of miR-29c-5p and screen out the targets that participated in regulating the Wnt pathway. (c) The binding of miR-29c-5p to LRP6 was proved by dual-luciferase reporter gene assay. (d) miR-29c-5p mimic or NC-mimic was transfected into astrocytes. QRT-PCR was carried out to quantify the LRP6 expression. (e) A total of 20 patients with ischemic stroke and 20 matched healthy controls were included, and the blood samples were isolated from them. The expression of LRP6 was analyzed by qRT-PCR. Data are presented as an average of three independent assays. ***P < 0.001 vs NC mimic.

Figure 6

Verification of the regulatory function of the miR-29c-5p/LRP6 axis in the TJP destruction of BMECs and inflammation caused by OGD-treated astrocytes: (a) Astrocytes transfected with miR-29c-5p inhibitor and si-LRP6 were treated with OGD for 3 h. The concentrations of IL-1β, IL-6, TNF-α, and TGF-β in the cell culture supernatant of astrocytes were measured by ELISA. (b) Western blot was carried out to test the protein levels of VEGF-A and MMP-9. (c) Astrocytes transfected with miR-29c-5p inhibitor and si-LRP6 were treated with OGD for 3 h and then cocultured with BMECs for 24 h. The protein levels of GFAP and GLP-1R were quantified using Western blot. (d) Analysis of the claudin-5, occludin, and ZO-1 protein levels using Western blot. Data are presented as an average of three independent assays. ***P < 0.001 vs NC-inhibitor + Si-NC. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs miR-29c-5p inhibitor + si-NC.