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. 2015 May 15;26(10):1786-96.
doi: 10.1091/mbc.E14-10-1474. Epub 2015 Mar 18.

MiR-34a regulates blood-tumor barrier function by targeting protein kinase Cε

Wei Zhao  1 , Ping Wang  1 , Jun Ma  1 , Yun-Hui Liu  2 , Zhen Li  2 , Zhi-Qing Li  1 , Zhen-Hua Wang  3 , Liang-Yu Chen  2 , Yi-Xue Xue  4
Affiliations

MiR-34a regulates blood-tumor barrier function by targeting protein kinase Cε

Wei Zhao et al. Mol Biol Cell. .

Abstract

MicroRNA-34a (miR-34a) functions to regulate protein expression at the posttranscriptional level by binding the 3' UTR of target genes and regulates functions of vascular endothelial cells. However, the role of miR-34a in regulating blood-tumor barrier (BTB) permeability remains unknown. In this study, we show that miR-34a overexpression leads to significantly increased permeability of BTB, whereas miR-34a silencing reduces the permeability of the BTB. In addition, miR-34a overexpression significantly down-regulates the expression and distribution of tight junction-related proteins in glioma endothelial cells (GECs), paralleled by protein kinase Cε (PKCε) reduction. Moreover, luciferase reporter gene analysis shows that PKCε is the target gene of miR-34a. We also show that cotransfection of miR-34a and PKCε inversely coregulates BTB permeability and protein expression levels of tight junction-related proteins. Pretreatment of ψεRACK, a PKCε-specific activator, decreases BTB permeability in miR-34a-overexpressed GECs and up-regulates expression levels of tight junction proteins. In contrast, pretreatment of εV1-2, a specific PKCε inhibitor, gives opposite results. Collectively, our findings indicate that miR-34a regulates BTB function by targeting PKCε; after phosphorylation, PKCε is activated and contributes to regulation of the expression of tight junction-related proteins, ultimately altering BTB permeability.

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Figures

FIGURE 1:
FIGURE 1:
Relative expression of miR-34a in ECs and GECs. The expression of miR-34a was detected by quantitative real-time PCR. Relative gene expression was normalized to U6, and analysis was performed by the 2−ΔΔCt method. Data represent means ± SD (n = 6). **p < 0.01 vs. EC group.
FIGURE 2:
FIGURE 2:
The permeability of BTB was altered after overexpression or silencing of miR-34a. (A) Relative expression of miR-34a after transfection with overexpression and silencing plasmids. The endogenous expression of miR-34a was detected by quantitative real-time PCR. The relative gene expression was normalized to U6, and analysis was performed by the 2−ΔΔCt method. Data represent means ± SD (n = 6). (B) TEER values of GECs expressed as Ω⋅cm2. Data represent means ± SD (n = 5). (C) HRP flux was calculated as pmol/cm2. Data represent means ± SD (n = 5). *p < 0.05 and **p < 0.01 vs. miR-34a (+) NC group; #p < 0.05 and ##p < 0.01 vs. miR-34a (–) NC group.
FIGURE 3:
FIGURE 3:
The protein expression of tight junction–related proteins was altered in GECs after overexpression or silencing of miR-34a. (A) Protein expression levels of ZO-1, occludin, and claudin-5 as assessed by Western blot. The relative IDVs of ZO-1, occludin, and claudin-5 are shown using GAPDH as an endogenous control. Values represent the means ± SD (n = 5, each group). **p < 0.01 vs. miR-34a (+) NC group; ##p < 0.01 vs. miR-34a (–) NC group. (B) The distribution and expression of tight junction-related proteins as detected by immunofluorescence assay. ZO-1, occludin, and claudin-5 were respectively labeled with fluorescent secondary antibody, and nuclei (blue) were labeled with DAPI. Scale bars, 20 μm.
FIGURE 4:
FIGURE 4:
The relative expression of PKCε in ECs and GECs. (A) mRNA expression of PKCε as detected by RT-PCR. (B) Protein expression of p-PKCε or PKCε as detected by Western blot assay. GAPDH was used as inner control. Data represent means ± SD (n = 5). **p < 0.01 vs. EC group.
FIGURE 5:
FIGURE 5:
Overexpression or silencing of miR-34a significantly affected the protein expression of p-PKCε and PKCε. (A) PKCε mRNA expression levels as assessed by RT-PCR. IDVs are shown using GAPDH as endogenous control. (B) p-PKCε and PKCε protein levels as assessed by Western blot. (C) Relative IDVs of p-PKCε/GAPDH and PKCε/GAPDH by Western blot. (D) Relative IDV of p-PKCε/PKCε by Western blot. IDVs are shown with GAPDH as endogenous control. Values represent the means ± SD (n = 5). **p < 0.01 vs. miR-34a (+) NC group; ##p < 0.01 vs. miR-34a (–) NC group.
FIGURE 6:
FIGURE 6:
PKCε is a direct target of miR-34a. (A) The putative binding site of PKCε 3′UTR matching with the seed region of miR-34a was predicted with the help of TargetScan. (B) Relative activity of LUC was expressed as firefly/renilla luciferase activity. Values are means ± SD (n = 6). **p < 0.01 vs. PKCε wt + miR-34a (+) NC group.
FIGURE 7:
FIGURE 7:
The permeability of BTB was inversely coregulated by miR-34a and PKCε in GECs. (A) TEER values of GECs were expressed as Ω⋅cm2. (B) HRP flux was calculated as pmol/cm2. Data represent means ± SD (n = 5). *p < 0.05 and **p < 0.01 vs. miR-34a (+) + PKCε (+) group; #p < 0.05 and ##p < 0.01 vs. miR-34a (–) + PKCε (+) group; ▴p < 0.01 vs. miR-34a (+) NC + PKCε (+) NC group; ▵p < 0.05 vs. miR-34a (–) NC + PKCε (–) NC group.
FIGURE 8:
FIGURE 8:
The expression levels of tight junction–related proteins were inversely coregulated by miR-34a and PKCε in GECs. (A, B) Protein expression levels of ZO-1, occludin, and claudin-5 as determined by Western blot. IDVs are shown with GAPDH as endogenous control. Data represent the means ± SD (n = 5). **p < 0.01 vs. miR-34a (+) + PKCε (+) group; ##p < 0.01 vs. miR-34a (–) + PKCε (+) group; ▴p < 0.01 vs. miR-34a (+) NC + PKCε (+) NC group; ▵p < 0.05 vs. miR-34a (–) NC + PKCε (-) NC group.
FIGURE 9.
FIGURE 9.
The permeability of BTB was regulated by PKCε activator ψεRACK and inhibitor εV1-2 peptides (100 nM). (A) TEER values of GECs expressed as Ω⋅cm2. (B) HRP flux calculated as pmol/cm2. Data represent means ± SD (n = 5). ▴p < 0.01 vs. control group; **p < 0.01 vs. miR-34a (+) group; ##p < 0.01 vs. miR-34a (–) group.
FIGURE 10:
FIGURE 10:
The expression of p-PKCε and PKCε, as well as of tight junction–related proteins, in GECs after pretreatment with PKCε activator ψεRACK or inhibitor εV1-2 (100 nM). (A) p-PKCε, PKCε, ZO-1, occludin, and claudin-5 protein levels as assessed by Western blot. (B) Relative IDVs of p-PKCε/GAPDH and PKCε/GAPDH as detected by Western blot. (C) Relative IDV of p-PKCε/PKCε as assessed by Western blot. (D) Relative IDVs of ZO-1 and occludin as determined by Western blot. IDVs are shown with GAPDH as endogenous control. Values represent the means ± SD (n = 5). ▴p < 0.01 vs. control group; *p < 0.05 and **p < 0.01 vs. miR-34a (+) group; ##p < 0.01 vs. miR-34a (–) group.
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