MiR-34a regulates blood–brain barrier permeability and mitochondrial function by targeting cytochrome c

BBB permeability assay in vitro

Cell culture and transfection were detailed in supplementary information (SI). To identify the effect of miR-34a on the BBB, we employed a CEC (bEnd.3 cell line from ATCC) culture model, which is a murine CEC line with established BBB characteristics. We first transfected the CECs with miRNA-Alxea-Fluo®-555 by lipofectomine RNAiMAX reagent and confirmed high transfection efficiency (74.9%) in CECs by flow cytometry (Supplementary Figure 1(a)) and microscopy (Supplementary Figure 1(b)). Then, we constructed a miR-34a expression plasmid and observed an approximate nine-fold increase in miR-34a copies in CECs with the miR-34a plasmid compared to vector control as determined by real-time polymerase chain reaction (PCR) (Supplementary Figure 1(c)). We performed the following experiments under the same transfection condition.

We transfected the CECs in 24-well plates and conducted BBB permeability assays in transwell inserts (Millipore, Darmstadt, Germany) in triplicates. We seeded transfected CECs (1.5 × 10⁵) on inserts for two days. We added 250 µg/ml FITC-labeled dextran FD-4 (4 kDa, Sigma) to the apical side of the filters and then sampled the medium in the basolateral compartment every 15 min for 2 h. We read the mean fluorescent intensity of FD-4 on a plate reader (excitation 490 nm and emission 515 nm) and calculated the apparent permeability coefficient (Papp, cm/s) with an FD-4 standard curve.

Oxygen consumption rate evaluation

We measured oxygen consumption rate (OCR) at 37℃ using an XF96e extracellular analyzer (Seahorse Bioscience, Massachusetts, USA) according to the manufacturer’s instructions. Briefly, we seeded CECs into Seahorse Bioscience XF96e cell culture plates (16,000 cells/well for 48 h culture; 10,000 cells/well for 72 h culture) and cultured overnight. We performed miR-34 plasmid versus vector control transfection with Lipofectamine RNAiMAX reagent (Life Technologies, New York, USA) and cultured CECs for additional 48 h or 72 h. We changed the cell culture medium to un-buffered (pH 7.4) DMEM prior to measurements. We prepared 10 × dilutions of oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone, and antimycin A (all from Sigma) and implemented the measure protocol. This protocol allowed determination of the basal level of oxygen consumption, the amount of oxygen consumption linked to adenosine triphosphate (ATP) production, maximal respiration capacity, and non-mitochondrial oxygen consumption.

ATP measurement

We cultured cells in 96-well black plates and measured ATP level by ATP bioluminescence assay kit (Promega, Wisconsin, USA) according to the manufacturer’s instruction.

Detect respiratory chain complex I–IV by flow cytometry

We cultured CECs in six-well plates and transfected the cells with miR-34a plasmid or control, then we washed the cells and performed intracellular staining with an intracellular staining set (cat. 72-5775, eBioscience, California, USA). Antibody information is detailed in SI. We acquired data on BD Calibur flow cytometry and analyzed mean fluorescence intensity by Flowjo software.

Statistical analysis

We performed statistical analysis using Prism 5 software (Graphpad software, California, USA). Differences between groups were analyzed by the unpaired Student’s t-test, 1-way analysis of variance (ANOVA), or 2-way ANOVA as indicated in the figure legends.

Overexpression of miR-34a affects BBB permeability and disrupts tight junctions in CECs

To determine whether miR-34a functionally affected the BBB, we transfected CECs with miR34a plasmid versus vector control in 24-well plates, cultured the cells for 48 h, conducted a BBB permeability assay in a CEC monolayer transwell system in vitro with an additional culture of 48 h, and measured the fluorescent dye FD-4 permeability of each well (Figure 1(a)). As shown in Figure 1(a), FD-4 permeability was significantly increased in wells containing miR-34a overexpression CEC monolayer. Papp, the permeability coefficient, was also significantly higher in CECs overexpressed with miR-34a in comparison to vector controls (Figure 1(a)). Furthermore, immunohis-tochemistry staining of tight junction-related proteins revealed that ZO-1 was continuously distributed in the control, but a discontinuous distribution of ZO-1 was observed in miR-34a overexpressed CEC monolayer (Figure 1(b)). Disruption of tight junctions was not associated with cell viability in CECs transfected with plasmids for 48 h or 96 h (Supplementary Figure 2). Altogether, these data suggest that overexpression of miR-34a increases BBB permeability and compromises BBB tight junctions.

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Figure 1.

Overexpression of miR-34a increases BBB permeability in vitro. (a) A schematic protocol using fluorescein isothiocyanate–dextran-4 (FD-4) to detect BBB permeability in vitro. FD-4 permeability in CECs that overexpressed miR-34a plasmid (0.017 ng) versus control was presented as real-time rate of FD-4 mean fluorescent intensity (2-way ANOVA followed by post hoc Dunnett’s test; n = 3; **, P < 0.01; ****, P < 0.0001). Calculated apparent permeability coefficient P_app (Student’s t-test; ****, P < 0.0001) is expressed as mean ± SD. (b) Confocal fluorescence images of CECs confluent monolayers confirmed microscopically after transfection with miR-34a plasmid versus control. Fluorescent staining: tight junctions ZO-1 (red), cell nuclei (DAPI, blue). Overexpression of miR-34a apparently disrupted tight junctions and resulted in gaps between cells (white arrows). Results are representative of three independent experiments.

MiR-34a affects mitochondrial function by targeting CYC in CECs

Our recent work demonstrated that mitochondria play a pivotal role in the maintenance of BBB integrity. BBB tight junctions are rapidly disrupted if oxidative phosphorylation is reduced by mitochondrial inhibitors. ⁴ To investigate whether the miR-34a regulates BBB openings via affecting mitochondrial function in CECs, we examined cellular energetic OCRs in CECs transfected with miR-34a plasmid versus vector control. Interestingly, overexpression of miR-34a significantly impaired mitochondrial function in CECs (Figure 2(a) and Supplementary Figure 3). Basal respiration, ATP production, maximal respiration, and spare capacity were all significantly reduced in CECs overexpressing miR-34a for 48 and 72 h (Figure 2(a)). ATP level was also substantially reduced in CECs following overexpression of miR-34a in a dose dependent manner at 72 h (Figure 2(b)).

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Figure 2.

Overexpression of mir-34a reduces mitochondrial function and decreases CYC level in cerebrovascular endothelial cells. (a) Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the bioenergetics functional assay at post-transfection 48 and 72 h (raw data in Supplementary Figure 3). Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (b) ATP level was measured at 72 h post-transfection. Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (****, P < 0.0001). (c) Bioinfomatic analysis of miR-34a-targeting candidates related to mitochondria. (d) Flow cytometry analysis of mitochondrial specific proteins for complex I proteins (NDUFAF1, NDUFC2 and NDUFS2), complex II protein (SDHC), complex III protein (CYB), complex IV protein (CYC oxidase, Cox IV), cytochrome c (CYCS), pyruvate dehydrogenase kinase (PDK), and voltage-dependent anion channel protein (VDAC) at 72 h post-transfection. CYC level was significantly lower in the cells that were transfected with the miR-34a plasmid. Data are presented as mean ± SD (n = 3) and analyzed by Student’s t-test, *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Results are representative of three independent experiments.

To further determine miR-34a targets and uncover the mechanism that is used to affect mitochondria, we performed a bioinformatics analysis of the miR-34a database (miRbase and TargetScan). MiR-34a potentially targets several mitochondria-associated gene candidates including succinate dehydrogenase subunit c (SDHC), cytochrome B reductase 1 (CYBRD1), cytochrome B5 reductase 3 (CYBRD5), cytochrome c (CYCS), pyruvate dehydrogenase kinase isozyme 1 and 2 (PDK1 and PDK2) (Figure 2(c). However, CECs transfected with the miR-34a plasmid had robustly decreased CYCS levels measured by flow cytometry, suggesting that CYCS is one of the miR-34a targets among the potential candidates (Figure 2(d)). Moreover, overexpression of miR-34a slightly increased potential target SDHC but did not change the protein level of CYB and PKD (Figure 2(d)). Off-target genes, NDUFAF1, and VDAC showed no significant change in protein level, but NDUFC2, NDUFS2, and Cox IV were all increased in parallel with overexpression of miR-34a (Figure 2(d)). Taken together, these results experimentally verified CYCS as a miR-34a target, which is associated with the reduction of mitochondrial oxidative phosphorylation in CECs.