Open Access

BIRC5 is a novel target of peroxisome proliferator‑activated receptor γ in brain microvascular endothelium cells during cerebral ischemia

  • Authors:
    • Mingjing Xu
    • Xianli Yang
    • Qing Zeng
    • He He
    • Pengcheng Lu
    • Guozhi Huang
  • View Affiliations

  • Published online on: October 10, 2017
  • Pages:8882-8890
  • Copyright: © Xu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Cerebral ischemia is a leading cause of ischemic stroke, which may lead to severe disability and mortality worldwide. There are some key factors concerned in cardioprotection, such as peroxisome proliferator‑activated receptor γ (PPARγ), a ligand binding transcription factor involved in various biological functions including atherosclerosis, vascular dysfunction and hypertension, and baculoviral IAP repeat‑containing 5 (BIRC5), which may protect human brain endothelial cells from ischemia‑induced apoptosis. To determine the potential roles of PPARγ in brain microvascular endothelial (bEnd.3) cells during cerebral ischemia and the relationship between PPARγ and BIRC5, a cerebral ischemia model was established with bEnd.3 cells cells by oxygen‑glucose deprivation (OGD) treatment. OGD treatment reduced proliferation and enhanced apoptosis of bEnd.3 cells in a time‑dependent manner. PPARγ expression levels were decreased in bEnd.3 cells following OGD treatment. Upregulation of PPARγ expression protected bEnd.3 cells from ischemia injury and also upregulated BIRC5 expression. PPARγ‑specific binding sites in the BIRC5 promoter were predicted bioinformatically and verified by luciferase reporter experiments. Results from electrophoretic mobility shift/supershift and chromatin immunoprecipitation assays suggested that BIRC5 may be a novel target of PPARγ transcriptional regulation during ischemic injury. The present results indicated that PPARγ may serve a protective role on bEnd.3 cells and that BIRC5 may be a downstream target of PPARγ regulation during cerebral ischemia.


Cerebral ischemia leads to insufficient oxygen supply and ischemic stroke (1,2), and is associated with a number of diseases or disorders (3). Microvascular endothelial cells can be activated during the hypoxia or ischemia, and by upregulating the expression levels of various agents, including proinflammatory mediators and adhesion molecules (4). Currently, there is no effective treatment or prevention available for the management of cerebral ischemia, and our knowledge is limited pertaining to brain microvascular endothelial (bEnd.3) cells during cerebral ischemia.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors with three distinct isoforms (PPARα, PPARγ and PPARδ) (5,6). PPAR activation serves a role in anti-inflammatory effects in the brain and may serve as a novel pharmacological target for the management of neurological diseases (7,8). Previous studies have revealed that brain ischemic injury enhanced the expression and activity of PPARγ, and PPARγ agonists may protect neuron cells against brain ischemic injury (9,10). However, the function of PPARs in bEnd.3 cells during cerebral ischemia remains unknown. Baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin) belongs to the inhibitor of apoptosis (IAP) gene family that is widely expressed in cancer cells (11). Hypoxic preconditioning may protect brain endothelium from ischemia-induced apoptosis by Akt-dependent BIRC5 activation (12), which implied a potential connection between BIRC5 expression and human brain endothelium injury. BIRC5 was also reported to cooperate with PARP proteins in studies on cell cycle (13) or on cell proliferation in bladder cancer cells (14). In short, the potential role of BIRC5 in cerebral ischemia and its interaction with PPARγ need to be elucidated (15).

The present study demonstrated that PPARγ may protect cerebral microvascular endothelium against ischemia-reperfusion injury, and that BIRC5 may be a novel target of PPARγ. These results may provide insights for future investigations considering the crucial role of PPAR regulators and targets in the pathogenesis of stroke.

Materials and methods

Cell culture

Mouse bEnd.3 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were maintained as previously described (16). Briefly, Cells were cultured in RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and supplemented with 15% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 100 U/ml penicillin and 100 U/ml streptomycin (Ameresco, Inc., Framingham, MA, USA). Cells were grown in a humidified atmosphere of 5% CO2/95% air at 37°C. The growth medium was replaced each day; cells were plated onto 96-well plates or Petri dishes for further analysis.

Plasmid construction and transfection. The complete coding sequence of PPARγ ( was amplified and cloned into pcDNA4.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). The bEnd.3 cells (2×105 cells/well) were seeded into 24-well plates and then pcDNA4.1-PPARγ overexpression plasmid (7 µg/ml, experimental group) or empty pcDNA4.1 vector (7 µg/ml, control group) was transfected into bEnd.3 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. The working concentration of plasmid was determined by previously study (17). After incubated at 37°C for 24 h, cells that stably overexpressed PPARγ were selected and moved to new 24-well plates at a concentration of 2×105 cells/well, after incubated overnight at 37°C, cells were treated with oxygen-glucose deprivation (OGD) for 12 h to establish an ischemic cell model, then western blot analysis and immunofluorescence assay were used to measure the expression of PPARγ in these cells.

Preparation of OGD model

To mimic ischemic conditions in vitro, bEnd.3 cells (2×105 cells/ml) were exposed to OGD. Cell cultures were subjected to ischemia-like injury through OGD for 3, 6 and 12 h by placing cultures in a Forma Anaerobic Chamber (Thermo Fisher Scientific, Inc.) with an atmosphere of O2 tension <0.2% (5% CO2, 5% H2 and 90% N2) in a deoxygenated glucose-free balanced salt solution. Cultures were placed in a humidified incubator at 37°C. Cultured cells and media were harvested by trypsinization and re-suspended in PBS, and then centrifugation at 16,000 × g for 10 min at 4°C as previously described (18,19) at different time points for further experiments.

Proliferation assays

The proliferative ability of bEnd.3 cells was measured using the Cell Counting Kit-8 (CCK-8), according to the manufacturer's instructions. The bEnd.3 cells were seeded into 24-well plates at a concentration of 2×105 cells/well, and incubated overnight at 37°C. After treated with OGD for 0, 3, 6 or 12 h, CCK-8 solution (10 µl) was added to 96-well plates incubated at 37°C for 4 h in 5% CO2, and the absorbance of each well was detected using a microplate reader (Multiskan Spectrum; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at a wavelength of 450 nm.

The 5-ethynyl-29-deoxyuridine (EdU) Cell-Light Apollo DNA in vitro Imaging kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China) was also used to examine proliferative ability. Cells (1×105 cells/dish) were cultured in Petri dishes for 24 h at 37°C. Following OGD treatment, 50 µM of EdU was added to each dish and cells were cultured for an additional 2 h at 37°C, and then cells stained with EdU were analyzed using the CellQuest Flow Cytometry System version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA).

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

The assay of qRT-PCR was performed as previously described (20). Briefly, total RNA was extracted from the cultured bEnd.3 cells (2×105 cells/ml) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. RNA was reverse transcribed using, and qPCR was performed with an ABI 9700 PCR Thermal Cycler and an SYBR-Green kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions. Briefly, qRT-PCR was performed using 10 µP 2X SYBR-Green PCR Master Mix (Toyobo Life Science, Osaka, Japan), with 5 µl of cDNA, 0.5 µl of primers and 4 µN of RNase-free ddH2O contained in 20 µl of reaction mixture. The reaction was performed with one cycle of 95°C for 5 min and 40 cycles of 95°C for 15 sec, 65°C for 15 sec and 72°C for 35 sec in ABI 7300 real-time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). When the reaction proceeded. Ct value was obtained, and results were analyzed using 2−∆∆Cq calculation (20). β-actin was used to normalize the data. The primer sequences were: β-actin forward, 5′-CATTGCTGACAGGATGCAGA-3′ and reverse, 5′-CTGCTGGAAGGTGGACAGTGA-3′; PPARγ forward, 5′-GGAAGACCACTCGCATTCCTT-3′ and reverse, 5′-GTAATCAGCAACCATTGGGTCA-3′; BIRC5 forward, 5′-GAGGCTGGCTTCATCCACTG-3′ and reverse, 5′-ATGCTCCTCTATCGGGTTGTC-3′. β-actin was used as an internal control.

Apoptosis assay

Apoptotic rates were examined by flow cytometric analysis using Annexin V staining kit (BD Pharmingen; BD Biosciences). The transfected bEnd.3 cells (1×106 cells/ml) or untransfected bEnd.3 cells (1×106 cells/ml) were collected by trypsinization and the suspensions centrifuged at 16,000 × g for 10 min at 4°C. Cells (1×106 cells/ml) were resuspended in 1X binding buffer (BD Biosciences). Subsequently, 100 µl of this solution (~1×105 cells) was transferred to a 5-ml culture tube. Annexin V (5 µl) and propidium iodide (5 µl; BD Biosciences), used for apoptosis signal detection, were added to the samples, and then incubated for 15 min at room temperature in the dark. A total of 400 ml 1X binding buffer was added to each tube and the samples were immediately analyzed by BD FACSCanto II flow cytometry (BD Biosciences). The data were analyzed by FlowJo software version 8.8.6 (FlowJo LLC, Ashland, OR, USA). For the Hoechst staining, treated or control cells were seeded in 24-well plates at a concentration of 1×106 cells/well and incubated overnight at 37°C, and the DNA content of cells in each well were stained with 100 µl of Hoechst 33342 for 30 min followed by DAPI staining for 10 min at room temperature and visualized under a fluorescence microscope (Olympus, Tokyo, Japan). Experiments were performed in triplicate.

Western blotting

Cell samples (2×106 cells/ml) were lysed in 4°C for at least 30 min by radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) to obtain total cell lysates. Protein concentrations were determined using the Bicinchoninic Acid Protein assay kit (Thermo Fisher Scientific, Inc.). Similar amounts of protein (40 µg) from each sample were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany). Membranes were incubated with primary rabbit polyclonal antibodies against PPARγ (ab59256, 1:1,000) and BIRC5 (ab469, 1:1500) (both from Abcam, Cambridge, MA, USA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated monoclonal goat anti-rabbit immunoglobulin (Ig)G (BA1054, 1:2,000; Boster Biological Technology, Pleasanton, CA, USA) at room temperature for 2 h. Membranes were stripped and reprobed with a primary monoclonal mouse anti-rabbit antibody against GAPDH (KF703, 1:1,000; Nanjing Jiancheng Bioengineering Institute, Nanjing, China,). Protein bands were quantified by densitometry using the gel analysis software ImageJ (National Institutes of Health, Bethesda, MD, USA).

Dual-luciferase reporter assay

The BIRC5 promoter binding site sequence (gene ID, 11799) for PPARγ was predicted using Ensembl ( and NCBI ( Wild-type (WT) BIRC5 promoter and a mutated (MUT) promoter sequence containing the predicted target sites were synthesized and cloned into the XbaI and FseI restriction sites of a pGL3 control vector (Promega, Madison, WI, USA); the constructs were termed pGL3-promoter-WT and pGL3-promoter-MUT. In the reporter assay experiment, bEnd.3 cells (1×103 cells/well) were seeded onto 24-well plates and transfected with either pGL3-promoter-WT or pGL3-promoter-MUT, and co-transfected with the pcDNA4.1-PPARγ or pcDNA4.1 control vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's instructions at 4°C for 2 h. A Renilla luciferase vector, pRL-SV50 (Promega), was also co-transfected into the cells and used to normalize the differences in firefly luciferase activities. Following 48 h transfection, cells were harvested and luciferase activities were measured with the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions. Transfections were repeated in triplicate in three independent experiments.

Immunofluorescence assay

PPARγ protein expression levels were also evaluated by immunofluorescence. Treated bEnd.3 cells (1×105 cells/ml) were seeded into 6-well plates overnight at 37°C. Then, the cells were fixed by 4% paraformaldehyde for 24 h, and blocked with 1% bovine serum albumin for 2 h at room temperature and incubated with anti-PPARγ antibody (cat. no. ab209350, 1:200; Abcam, Cambridge, UK) overnight at 4°C and then incubated with 1 µg/ml DAPI dihydrochloride; D9542; Sigma-Aldrich; Merck KGaA) at room temperature for 10 min. Fluorescence images of six random fields were captured on a Nikon Eclipse Ti-U fluorescence microscope (Nikon, Tokyo, Japan) equipped with a SPOT-RTTM digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). The fluorescent images were visualized with a Leica fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). Each experimental was replicated three times.

Electrophoretic mobility shift assay (EMSA) and supershift assay

Nuclear proteins from bEnd.3 cells (1×106 cells/ml) were extracted using the NE-PER® Nuclear Extraction Reagent (Thermo Fisher Scientific, Inc.) according to a previously described protocol (21). The treated cells were washed for three times with cold phosphate-buffered saline (PBS; pH 7.4), the cells were then scraped and centrifuged at 15,000 × g for 5 min, to collect the pellets. Biotin-labeled PPARγ specific oligonucleotides (Invitrogen; Thermo Fisher Scientific, Inc.), with the following sequence, 5′-AAAGGAGGTTAGAGGGGAAGGGGCGTAG-′3, were prepared as labeled probes, according to the manufacturer's instructions (Promega). Double-stranded oligonucleotides for PPARγ were end-labeled with adenosine-5′-triphosphate (ATP)-γ-32P using the T4 polynucleotide kinase (Promega), according to the manufacturer's instructions. Biotin end-labeled double-stranded DNA and the nuclear extracts were incubated at room temperature for 20 min, and then 10 µl protein-DNA complex was subjected to 6.5% PAGE at 100 V for 1 h at 4°C and transferred onto a nylon membrane. The radiolabeled probes were purified by spin columns (Roche Applied Science, Penzberg, Germany). Nuclear protein extracts (5 µg) from bEnd.3 cells were incubated with 100,000 cpm 32P-labeled oligonucleotide probe in 25 mM HEPES (pH 7.4), 50 mM KCl, 10% glycerol (v/v), 5 mM dithiothreitol and 1 µg of poly (deoxyinosinic-deoxycytidylic) acid (GE Healthcare Life Sciences, Shanghai, China) for 30 min at room temperature in a final volume of 20 µl. Following binding, protein-DNA complexes were separated on a 6% non-denaturing polyacrylamide gel at 120 V in 0.5X Tris/borate/EDTA buffer. Gels were analyzed with a PhosphorImager Gel Imaging System (Bio-Rad Laboratories, Inc.). For the antibody supershift analysis, 1 µg of antibody against PPARγ (ab59256; Abcam) was added to the nuclear extracts at a dilution of 1:1,000 for 16 h prior to addition of the radiolabeled oligonucleotides.

Chromatin immunoprecipitation (ChIP) assay

Cells (5×107) were fixed with 4% paraformaldehyde for 10 min at 37°C and a ChIP assay was performed with the EZ-ChIP assay kit (Sigma-Aldrich; Merck KGaA), as previously described (22). Briefly, Pellet cells were resuspended by SDS lysis buffer with 1% protease inhibitor cocktail set III EDTA-free (Calbiochem; Merck KGaA) and incubated for 10 min on ice. Sonicate lysate to shear DNA to lengths between 500–1,000 bp which were detected by 1% ethidium bromide gel electrophoresis. The conditions have been optimized following steps number of bursts: 8, length of bursts: 10 sec, interval time: 10 sec, output control setting: 30%, duty cycle: constant. The lysates were incubated with anti-PPARγ antibody (ab59256, 1:10; Abcam) or a rabbit control IgG (ab6789, 1:500; Abcam) for 24 h at 4°C, and the complexes were isolated using protein A-agarose/salmon sperm DNA (EMD Millipore, Billerica, MA, USA). Immunoprecipitates were added in 1 ml of high-salt wash buffer for ChIP to all samples and rotated for 10 min at room temperature; the samples were centrifuged at 4500 × g for 2 min at room temperature; the supernatants were carefully aspirated and added 1 ml of high-salt wash buffer for ChIP; the samples were rotated for 10 min at room temperature; the above two steps were repeated twice in a total of four high-salt washes; the supernatants were aspirated and washed twice with TE as above. Immunoprecipitates were subsequently eluted with freshly prepared 1% SDS + 0.1 M NaHCO3 buffer. To the immune complexes were added 20 µl 5 M NaCl and histone-DNA crosslinks reversed by heating at 65°C for 4 h. Following the reversing of crosslinking, DNA was purified with the QIAquick PCR Purification kit (Qiagen GmbH, Hilden, Germany) followed by PCR amplification. The amplification reactions were performed using Amplitaq DNA polymerase, GeneAmp dNTPs (deoxynucleoside triphosphates) with dUTP, and AmpErase UNG (all from PerkinElmer, Inc., Waltham, MA, USA). The thermocycling conditions were a thermal cycler preheated to 94°C; and then 94°C for 1 min, 55°C for 1 min, 72°C for 2 min for 30 cycles and a final extension at 72°C for 7 min. The PCR-amplified products were examined by electrophoresis in a 1.5% agarose gel, stained with a 1% solution of ethidium bromide, and examined under ultraviolet illumination. Primers used to amplify the PPARγ binding site area were: Forward, 5′-TCCCTTCCAACCTCCCAAT-3′ and reverse, 5′-AGCCCAGTATCCCAAATCAAC-3′, which resulted in a 98 bp fragment. PCR products were resolved by 2% agarose gel electrophoresis, visualized by ethidium bromide staining and analyzed by densitometry using ImageJ software version 1.37 (National Institutes of Health, Bethesda, MD, USA). To ensure the specificity of each assay, DNA binding in normal IgG immunoprecipitates was regarded as the background control.

Statistical analysis

All experiments were performed at least three times, and all samples were tested in triplicate. Experimental data are displayed as the mean ± standard deviation. All analyses were performed using one-way analysis of variance or an unpaired Student's t-test performed on SPSS software, version 12.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.


OGD reduces proliferation and enhances apoptosis of bEnd.3 cells

To establish a cerebral microvascular endothelial cell injury model with cerebral ischemia, bEnd.3 cells were exposed to OGD conditions to mimic ischemia-like conditions in vitro. Following OGD treatment, bEnd.3 cells exhibited a significant decrease in proliferation capacity in a time-dependent manner (Fig. 1A), with the group treated for 12 h exhibiting ~60% loss in cell viability compared with the control group (treated for 0 h). To confirm cell viability, the proliferation capacity of bEnd.3 cells post-treatment was examined by EdU incorporation and flow cytometry; the M2 phase rate was 11.9%, much lower compared with the control group (36.7%; Fig. 1B). The results demonstrated that viability in bEnd.3 cells significantly decreased following OGD treatment in a time-dependent manner.

The effects of OGD treatment on apoptosis in bEnd.3 cells were examined using the apoptosis assay by flow cytometry analysis and Annexin V/PI staining. OGD-treated cells exhibited an increase in the proportion of early apoptotic cells (5.3% in 3 h treatment group, 11.0% in 6 h treatment group and 16.0% in 12 h treatment group) and late apoptotic cells (3.1% in 3 h treatment group, 8.3% in 6 h treatment group, 9.5% in 12 h treatment group), compared with 4.5% (early) and 3.3% (late) in the 0 h control group (Fig. 1C), which resulted in an overall threefold enhancement in total cell apoptosis in the 12 h treatment group compared with the control (P<0.05; Fig. 1D). Following incubation and treatment, bEnd.3 cell nuclei were stained with Hoechst, and notable changes in cell apoptosis were observed, as the Hoechst nuclear staining became increasingly bright with the longer durations of OGD treatment (Fig. 1E).

PPARγ expression is inhibited by OGD treatment

To evaluate the status of PPARγ expression following OGD treatments of bEnd.3 cells, PPARγ mRNA and protein expression levels were detected. RT-qPCR analysis and western blotting revealed that the OGD treatment led to reduction in PPARγ mRNA and protein expression levels (Fig. 2A and B, respectively), and this decrease occurred in a time-dependent manner. To examine the PPARγ protein expression status in situ, immunofluorescence staining was performed (Fig. 2C). Untreated bEnd.3 cells (0 h control) exhibited uniform distribution of PPARγ expression compared with OGD-treated cells, which obtained exhibited a faint staining pattern. These results confirmed that PPARγ expression was inhibited in bEnd.3 cells treated with OGD.

PPARγ protects brain endothelium from ischemic apoptosis

A previous report suggested that PPARγ activation may protect neural cells following cerebral ischemia (23); therefore, the present study hypothesized that the upregulation of PPARγ gene expression may also relieve cerebral microvascular endothelial cells from cerebral ischemia injury. To examine this, bEnd.3 cells were transfected with pcDNA4.1-PPARγ overexpression plasmid, which exhibited a notable increase in PPARγ protein expression levels compared with cells transfected with the pcDNA4.1-empty vector control, in the presence or absence of OGD treatment (Fig. 3A). No significant difference was observed between bEnd.3 cells transfected with pcDNA4.1-PPARγ overexpression plasmid and pcDNA4.1-empty vector control without the treatment of OGD. However, in the presence of OGD treatment, the PPARγ expression was significantly higher in the PPARγ overexpressed bEnd.3 cells compared with the vector group (Fig. 3B).

PPARγ overexpression alleviates bEnd.3 cell death caused by 12 h OGD exposure

Results from live cell counts demonstrated that the cell viability recovered more than 50% in the bEnd.3 cells transfected with pcDNA4.1-PPARγ overexpression plasmid compared with those cells transfected with the empty vector (Fig. 4A). Proliferation capacity of bEnd.3 cells was also analyzed by EdU incorporation assay (Fig. 4B); M2 phase rate was notably higher (~17%) in OGD-treated cells that were transfected with the PPARγ overexpression plasmid compared with OGD-treated cells transfected with the empty vector (8.8%). No significant differences in viability were identified in bEnd.3 cells transfected with pcDNA4.1-PPARγ plasmid compared with the control groups (Fig. 4A and B). In the Annexin V/PI apoptosis assay, the proportion of total apoptotic cells was observed to be 7.2% in the control group, 4.7% in the PPARγ overexpression group, 29.5% in the OGD treatment group and 18.3% in the OGD-treated cells that overexpressed PPARγ (Fig. 4C and D), which indicated that PPARγ may significantly inhibit apoptosis under ischemia-like conditions. Hoechst nuclear staining also demonstrated that PPARγ overexpression may rescue bEnd.3 cell death following OGD treatment (Fig. 4E).

BIRC5 expression is regulated by PPARγ during ischemia

To further determine the molecular mechanisms responsible for PPARγ-mediated protective roles in the OGD treatment process, the expression of another critical factor that also serves important roles in ischemic apoptosis, BIRC5, was examined. BIRC5 mRNA expression was significantly decreased in bEnd.3 cells following OGD treatment, compared with control cells (Fig. 5A); this reduced expression was recovered in OGD-treated cells that overexpressed PPARγ. Similar results were obtained in western blot analyses of BIRC5 protein expression (Fig. 5B). These results indicated that BIRC5 expression may be regulated at both the mRNA and the protein level by PPARγ during ischemic conditions.

BIRC5 is a target of PPARγ regulation

To elucidate the mechanisms of PPARγ regulation on BIRC5, a functional analysis was performed to verify the potential PPARγ binding sites in the BIRC5 promoter. The transcriptional responses of the BIRC5 pGL-promoter-WT and pGL-promoter-MUT plasmids were analyzed using an in vitro luciferase transcriptional assay (2). pcDNA4.1-PPARγ overexpression significantly increased the transcriptional activity of the pGL-promoter-WT, compared with cells co-transfected with the empty pcDNA4.1 vector (Fig. 5C). Conversely, no significant differences were identified in the luciferase activities of the pGL-promoter-MUT group co-transfected with the PPARγ overexpression vector.

ChIP analysis was applied to verify the interaction between PPARγ and BIRC5 promoter. Consistent with previously reported transcriptional activity, a significant increase in PPARγ binding to the BIRC5 promoter site was detected, with isotypic IgG antibody used as a negative immunoprecipitation control (P<0.01; Fig. 5D).

EMSA supershift assay was applied to determine whether PPARγ was able to bind to the BIRC5 promoter. The PPARγ protein formed a complex band (shift band) using probes (Fig. 5E). By contrast, the PPARγ protein competitor prevented the formation of the shift band, which indicated that it interfered DNA binding. Specificity of binding was examined with a mutated competitor, which failed to elicit competition, as demonstrated in the unaltered band formation. The specificity of the complex was reconfirmed using a PPARγ antibody that supershifted the PPARγ + BIRC5 band. These data suggested that BIRC5 may be a novel target of PPARγ transcriptional regulation.


Cerebral microvascular endothelial cells serve a major role in ischemic insult of the brain, and regulate the trafficking of cells, substrates and other molecules across the blood-brain barrier, vasomotor reactivity and homeostasis at the interface of the blood/vascular wall. Neurovascular protection is considered as an effective part of stroke therapy (6,24). Elucidation of the underlying mechanism of different regulators and bEnd.3 cells may provide new insights into the cerebral vasculature and provide a novel therapeutic strategy for the treatment of diseases such as stroke.

Cell culture models of cerebrovascular endothelium are essential for exploring the molecular mechanisms of ischemic injury (18,2527). The present study established an OGD-induced apoptotic injury model using the mouse microvascular endothelial cell line bEnd.3 and found the most suitable duration time (12 h) of this model, which may facilitate the future study of ischemic injury. Similar to previous studies (3,12,28), the present study revealed that PPARγ protein expression was reduced in this established model. In order to further investigate the biological functions of PPARγ, a PPARγ overexpressed cell model was established by transfecting bEnd.3 cells with PPARγ overexpression plasmid. Cell proliferation and apoptosis assay demonstrated that PPARγ may act as a vascular protective agent in the OGD treated cell model. PARγ activation has been reported to stimulate proliferation and attenuate apoptosis in endothelial progenitor cells through PPARγ-dependent signaling cascades (8,29,30). Results from another study also indicated that PPARγ may inhibit H2O2-induced apoptosis of bEnd.3 cells by upregulating the expression level of 14-3-3 (5,10). It is hypothesized that PPARγ and its ligands may serve as protective agents in the brain during stroke (31).

BIRC5 is a known survival factor in studies on embryogenesis and oncology (11,32), and was recently revealed to serve an important role in cerebral microvascular endothelial cell injury (33,34). However, the regulatory function of PPARγ on BIRC5 was not widely recognized. The present study demonstrated that PPARγ upregulation regulated the expression of BIRC5 both transcriptionally and post-translationally in OGD-treated cells. In addition, the upregulation of BIRC5 may be involved in the acquired resistance from various of noxious stimulations, such as ischemia and other lesions (35). The present study was the first, to the best of the authors' knowledge, to reveal that BIRC5 can be regulated by PPARγ via directly binding in ischemia. In conclusion, the present results demonstrated a cerebrovascular protective role of PPARγ in an ischemia model and identified BIRC5 as a novel target in the pathogenesis of ischemic vascular injury. Therefore, pharmacological activation of either PPARγ or BIRC5 expression may provide potentially therapeutic options for vascular damage induced by ischemia in clinical treatment. The mechanisms of PPARγ-mediated protection in endothelial cell damage explained in this study may aid in further understanding the pathogenesis and therapy of cerebral ischemia.

Previous studies have demonstrated that IRC5 may be a pejorative prognostic marker in stage II/III breast cancer (36), that silencing of BIRC5 induces neuroblastoma apoptosis (37). BIRC5 is involved in the biological processes of colorectal cancer (38) and can serve as a serum diagnostic and prognostic biomarker of colorectal cancer (39). Silencing BIRC5 promotes hepatoma cell apoptosis (40). However, the expression level of BIRC5 in endothelial cells treated with OGD following PPARγ treatment and the relationship between BIRC5 and PPARγ remain to be elucidated. The present study identified that OGD treatment markedly decreased BIRC5 expression in bEnd.3 cells, and that overexpression of PPARγ recovered this reduction mediated by OGD. In addition, it was identified that PPARγ increased the transcriptional activity of BIRC5, which suggested that BIRC5 is a target of PPARγ regulation. ChIP assay also indicated that there was a significant increase in PPARγ binding to the BIRC5 promoter site, suggesting that PPARγ can interact with BIRC5 promoter. EMSA supershift assay further showed that PPARγ can bind to the BIRC5 promoter. Therefore, it was demonstrated that BIRC5 may be a novel target of PPARγ transcriptional regulation.



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December 2017
Volume 16 Issue 6

Print ISSN: 1791-2997
Online ISSN:1791-3004

2016 Impact Factor: 1.692
Ranked #19/128 Medicine Research and Experimental
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Xu, M., Yang, X., Zeng, Q., He, H., Lu, P., & Huang, G. (2017). BIRC5 is a novel target of peroxisome proliferator‑activated receptor γ in brain microvascular endothelium cells during cerebral ischemia. Molecular Medicine Reports, 16, 8882-8890.
Xu, M., Yang, X., Zeng, Q., He, H., Lu, P., Huang, G."BIRC5 is a novel target of peroxisome proliferator‑activated receptor γ in brain microvascular endothelium cells during cerebral ischemia". Molecular Medicine Reports 16.6 (2017): 8882-8890.
Xu, M., Yang, X., Zeng, Q., He, H., Lu, P., Huang, G."BIRC5 is a novel target of peroxisome proliferator‑activated receptor γ in brain microvascular endothelium cells during cerebral ischemia". Molecular Medicine Reports 16, no. 6 (2017): 8882-8890.