Hypoxia Inhibits Cell Cycle Progression and Cell Proliferation in Brain Microvascular Endothelial Cells via the miR-212-3p/MCM2 Axis

Abstract

Hypoxia impairs blood-brain barrier (BBB) structure and function, causing pathophysiological changes in the context of stroke and high-altitude brain edema. Brain microvascular endothelial cells (BMECs) are major structural and functional elements of the BBB, and their exact role in hypoxia remains unknown. Here, we first deciphered the molecular events that occur in BMECs under 24 h hypoxia by whole-transcriptome sequencing assay. We found that hypoxia inhibited BMEC cell cycle progression and proliferation and downregulated minichromosome maintenance complex component 2 (Mcm2) expression. Mcm2 overexpression attenuated the inhibition of cell cycle progression and proliferation caused by hypoxia. Then, we predicted the upstream miRNAs of MCM2 through TargetScan and miRanDa and selected miR-212-3p, whose expression was significantly increased under hypoxia. Moreover, the miR-212-3p inhibitor attenuated the inhibition of cell cycle progression and cell proliferation caused by hypoxia by regulating MCM2. Taken together, these results suggest that the miR-212-3p/MCM2 axis plays an important role in BMECs under hypoxia and provide a potential target for the treatment of BBB disorder-related cerebrovascular disease.

Keywords: MCM2; blood–brain barrier; cell cycle; hypoxia; miR-212-3p; proliferation.

Figure 1

Functional analysis of differentially expressed genes (DEGs) in bEnd.3 cells under 24 h hypoxia. (A) Volcano map of differentially expressed genes between the control and hypoxia conditions. (B) Heatmap of the top upregulated and downregulated genes. (C) Gene ontology (GO) term analysis of differentially expressed genes. (D) Top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. (E) Top 10 upregulated pathways from gene set enrichment analysis (GSEA). (F) Top 10 downregulated pathways from GSEA.

Figure 2

Hypoxia inhibits microvascular endothelial cell cycle progression and cell proliferation. (A) GSEA curves of differentially expressed genes for the hypoxia vs. control comparison. (B) The expression of cyclin−dependent kinase 1(Cdk1), Cdk2, Cdk4, cyclin A2, cyclin B1, cyclin D1, and cyclin E1 from RNA−seq data. (C) Western blot analysis of CDK1, CDK2, CDK4, cyclin A2, cyclin B1, cyclin D1, and cyclin E1 (n = 3). (D) The percentages of cells in the G0/G1, S, and G2/M phases of the cell cycle were quantified by flow cytometry (n = 3). (E) The EdU (red) assay was used to examine cell proliferation (n = 3). EVOS M5000 objective magnification: 40×. (F) Cell proliferation was evaluated by a CCK−8 assay (n = 3). (G) Proliferating cell nuclear antigen (PCNA) and osteopontin (OPN) expression was determined by Western blotting (n = 3). Data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01. Hyp indicates hypoxia, and Con indicates control.

Figure 3

Knockdown of Minichromosome maintenance complex component 2 (Mcm2) inhibits cell cycle progression and cell proliferation. (A) The expression of Mcm2 from RNA-seq data and qRT–PCR (n = 3). (B) MCM2 expression was determined by Western blotting (n = 3). (C) The EdU (red) assay was used to examine cell proliferation (n = 3). EVOS M5000 objective magnification: 20×. (D) Cell proliferation was evaluated by a CCK-8 assay (n = 3). The symbol * (red) represents the shMcm2 vs. NC-sh group. (E) Western blot analysis of MCM2, PCNA, OPN, CDK2 and cyclin A2 (n = 3). Data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01. NC indicates negative control, and OE indicates overexpression.

Figure 4

MCM2 attenuates the inhibition of cell cycle progression and cell proliferation caused by hypoxia. (A) The EdU (red) assay was used to examine cell proliferation (n = 3). EVOS M5000 objective magnification: 20×. (B) Cell proliferation was evaluated by a CCK-8 assay (n = 3). The symbol * (orange) represents the Hyp + Mcm2-OE vs. Hyp + NC-OE group. (C) Western blot analysis of Mcm2, PCNA, OPN, CDK2, and cyclin A2 (n = 3). Data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01.

Figure 5

miR-212-3p directly downregulates MCM2 in microvascular endothelial cells under hypoxia. (A) Volcano map of differentially expressed miRNAs between the control and hypoxia groups. (B) Heatmap of the top upregulated and downregulated miRNAs. (C) GO term analysis of target genes for differentially expressed miRNAs. (D) KEGG pathways analysis of target genes for differentially expressed miRNAs. (E) Venn diagram of significantly upregulated miRNAs targeting MCM2. (F) Expression of miR-212-3p from RNA-seq data and qRT–PCR (n = 3). (G) Schematic diagram of the predicted binding sequences of miR-212-3p to the MCM2 3’UTR WT or MUT. (H) Luciferase activity was detected by dual luciferase assay after cotransfection of MCM2 3′UTR-WT/MUT as well as miR-212 mimic and mimic NC in 293T cells. Data are shown as the mean ± SEM, ** p < 0.01.

Figure 6

miR-212-3p inhibitor attenuates the inhibition of cell cycle progression and cell proliferation caused by hypoxia. (A) The EdU (red) assay was used to examine cell proliferation (n = 3). EVOS M5000 objective magnification: 20×. (B) Cell proliferation was evaluated by a CCK-8 assay (n = 3). The symbol * (red) represents the mimic-212 vs. mimic-NC group. (C) Western blot analysis of MCM2, PCNA, OPN, CDK2, and cyclin A2 (n = 3). (D) The EdU (red) assay was used to examine cell proliferation (n = 3). EVOS M5000 objective magnification: 20×. (E) Cell proliferation was evaluated by a CCK-8 assay (n = 3). The symbol * (orange) represents Hyp + inhibitor-NC vs. Hyp + inhibitor-212. (F) Western blot analysis of MCM2, PCNA, OPN, CDK2, and cyclin A2 (n = 3). Data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01.

Figure 7

miR-212-3p attenuated hypoxia-induced cell cycle progression and cell proliferation inhibition by regulating MCM2. (A) The EdU (red) assay was used to examine cell proliferation (n = 3). EVOS M5000 objective magnification: 20×. (B,D) Western blot analysis of MCM2, PCNA, OPN, CDK2, and cyclin A2 (n = 3). (C) Cell proliferation was evaluated by a CCK-8 assay (n = 3). The symbol * (green) represents Hyp + inhibitor-212 + shMcm2 vs. Hyp + inhibitor-212 + NC-sh. Data are shown as the mean ± SEM, * p < 0.05, ** p < 0.01.