Clin Transl Oncol (2015) 17:41–49 DOI 10.1007/s12094-014-1195-5
RESEARCH ARTICLE
EZH2 promotes tumor progression by increasing VEGF expression in clear cell renal cell carcinoma Z. Q. Xu • L. Zhang • B. S. Gao • Y. G. Wan X. H. Zhang • B. Chen • Y. T. Wang • N. Sun • Y. W. Fu
•
Received: 31 March 2014 / Accepted: 11 June 2014 / Published online: 2 July 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Objectives The present study is to evaluate the expression level of enhancer of zeste homolog 2 (EZH2) and vascular endothelial growth factor (VEGF), and analyze their correlations with clinicopathological characteristics and survival in patients with clear cell renal cell carcinoma (CCRCC). The effect of EZH2 on apoptosis and cell proliferation in 786-O renal cancer cell line is investigated. Methods The expression level of EZH2 and VEGF was detected in 185 primary CCRCC patients’ tissues using tissue microarray and immunohistochemistry. Small interfering RNA or enhanced green fluorescent protein transfection was employed to investigate the effect of EZH2 inhibition or overexpression on VEGF expression, apoptosis and cell proliferation in 786-O cells using flow cytometry, immunofluorescence microscopy, quantitative real-time reverse-transcription polymerase chain reaction and Western blot analysis. Results High expression level of EZH2 and VEGF was observed in advanced CCRCC and correlated with the TNM stage (p = 0.013, p = 0.001) and distant metastasis
Z. Q. Xu B. S. Gao Y. T. Wang N. Sun Y. W. Fu (&) Urology Center, The First Hospital, Jilin University, Changchun 130021, Jilin, People’s Republic of China e-mail: [email protected] Z. Q. Xu e-mail: [email protected] Z. Q. Xu Y. G. Wan X. H. Zhang B. Chen Department of Urology, Tongliao Hospital, Tongliao 028000, Inner Mongolia, People’s Republic of China L. Zhang Department of Pharmacy, Tongliao Hospital, Tongliao 028000, Inner Mongolia, People’s Republic of China
(p = 0.011, p = 0.038), respectively. EZH2 was positively correlated with VEGF in CCRCC tissues (correlation coefficient = 0.850, p \ 0.001). Kaplan–Meier survival analysis revealed that patients with positive EZH2 expression had a shorter overall survival time compared to patients with negative EZH2 expression (34.3 vs. 67.2, p \ 0.001). In 786-O cells, EZH2 silencing inhibited VEGF expression and cell proliferation while increasing apoptosis (p \ 0.001). EZH2 overexpression promoted VEGF expression and cell proliferation while inhibiting apoptosis (p \ 0.001). Conclusions EZH2 correlates positively with VEGF and associates with adverse clinicopathologic characteristics and shorter survival time in CCRCC patients. EZH2 accelerates antiapoptosis and cell cycle in 786-O cells. Keywords EZH2 VEGF Apoptosis Proliferation Clear cell renal cell carcinoma
Introduction Renal cell carcinoma (RCC), comprising 90 % of primary malignant renal tumors in adults, originates from the renal parenchyma and remains a major cause of morbidity and mortality [1]. Clear cell renal cell carcinoma (CCRCC) is the major histological subtype of RCC, comprising around 71 % of all RCC cases [2]. Approximately, 40 % of CCRCC patients eventually die of cancer progression with the highest fatality rate among the common urologic malignancies. The enhancer of zeste homolog 2 (EZH2), located on chromosome 7q35, is a member of the polycomb-group (PcG) genes required for the stable transmission of gene expression patterns to progeny cells throughout
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development. It functions as a transcriptional regulator for the maintenance of cell cycle regulation, cell identity and oncogenesis. EZH2, embryonic ectoderm development (EED), Yin-Yang-1 (YY1) and suppressor of zeste 12 (SUZ12) constitute the polycomb repressive complex (PRC) 2 [3]. EZH2 serves as a histone methyltransferase and results in silencing of various genes, including tumor suppressor genes, through direct methylation of lysine-27 of histone H3. Dysregulated expression of EZH2 is associated with human hematologic malignancies, such as malignant myeloid disorders [4] and lymphomas [5, 6] as well as epithelial malignancies including prostate [7], bladder [8], and skin cancers [9]. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes angiogenesis in tumorigenesis [10]. VEGF has been implicated with tumor growth, metastasis and poor prognosis in renal cancer and anti-VEGF therapy has been widely used for the treatment of RCC [11]. Studies have shown that VEGF-mediated down-regulation of miR-101 results in increased expression of EZH2 in angiogenic endothelial cells and decreased expression of EZH2 inhibits vascularization in glioblastomas. These results suggest a possible therapeutic potential for EZH2 inhibition in tumors with aberrant vascularization [12]. EZH2 inhibition increases the apoptotic activity and accelerates the cell cycle in small cell lung carcinoma [13] and in patients with cholangiocarcinoma [14]. Although both EZH2 and VEGF have been correlated with poor prognosis of RCC, little is known about the role EZH2 plays in tumor angiogenesis and the effect of EZH2 on apoptosis and cell cycle regulation in CCRCC. The objective of this study is to determine the expressions of EZH2 and VEGF and their correlations with clinicopathological characteristics and survival in patients with CCRCC. We also explored the effect of EZH2 on apoptosis and cell proliferation in 786-O renal cancer cell line.
Materials and methods Patients’ parameters One hundred and eighty-five renal cancer patients treated in the Urology Center of the First Hospital, Jilin University in Changchun China from 2000 to 2013 were selected for this study. Written informed consents were obtained from all patients according to the rules of Jilin University Ethnics Committee. The inclusion criteria were: CCRCC and no radiotherapy or chemotherapy. The clinicopathological characteristics, defined by the World Health Organization (WHO) criteria and the seventh edition of the American Joint Committee of Cancer (AJCC) staging system [2, 15], included age, gender, histologic grade, TNM stage and
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distant metastasis (Table 1). Tissue specimens from these patients were collected from the Department of Pathology and the Urology Center in the First Hospital of Jilin University including 185 paraffin-embedded primary tumor samples. Normal kidney tissues, 5 cm to the tumor area were used as normal controls. The overall survival time of the patients after tumor removal was 58.5 months (range 16–98 months). At the time of censoring the data, the follow-up information of 48 (26 %) patients was lost due to death or other reasons. Tissue microarray and immunohistochemical staining Tissue microarray construction and immunohistochemical staining were performed as described previously [16, 17]. Briefly, tissue microarray slides were stained with antibodies to EZH2 (mouse anti-human polyclonal antibody, Cell Signaling Technology, Inc., Danvers, MA, USA) and VEGF (rabbit anti-human polyclonal antibody, Abcam, Cambridge, MA, USA) based on the standard streptavidin– biotin complex method. Prostate adenocarcinoma tissues were used as positive controls. Phosphate-buffered saline (PBS, pH = 7.4) replaced the primary antibody as a negative control. The samples were analyzed under a 409 objective lens of an Axio Imager Z2 microscope (Carl Ziess AG, Jena, Germany). Ten fields were randomly selected within three randomly chosen representative tissue cores of a specimen on the tissue microarray slide. One hundred tumor cells per patient were analyzed. Cells with dark brown particles in the nuclei or cytoplasm were regarded as having positive protein expression. The immunohistochemical staining was Table 1 patients
Clinical pathologic characteristics of clear cell RCC
Characteristics
No. of patients (N = 185) (%)
Age (year) B52
73
[52
112
Gender Male Female
105 80
Histologic grade Grade 1–2
77
Grade 3–4
108
TNM stage I–II
98
III–IV
87
Distant metastasis Absent
127
Present
58
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recorded as: no expression (absent staining = 0), low expression (C30 % of tumor cells with weak staining intensity = 1), or high expression (C30 % of tumor cells with strong staining intensity = 2). No expression was regarded as negative and low or high expression as positive.
A total of 100 ll of the mixture was added to 400 ll of culture medium in each well. Cells were incubated at 37 °C in a 5 % CO2 incubator for 72 h and then tested for transgene expression.
Cell line and culture
Apoptosis in EZH2 silencing or overexpressed 786-O cells was quantified using flow cytometry. Cells were harvested and pooled after 72 h transfection and stained with Annexin V-PE (BD Biosciences, San Jose, CA, USA), a marker of early phase apoptosis, and with 7-aminoactinomycin (7AAD) (BD Biosciences, San Jose, CA, USA), an indicator of late apoptosis. Briefly, cells were rinsed twice in PBS and suspended in binding buffer containing 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/ NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Staining mixture of 5 ll Annexin V-PE and 5 ll 7-AAD was added to 100 ll of cell suspension (1 9 106 cells/ml). Cells were incubated in the dark for 15 min at room temperature and then analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Percentage of apoptotic cells that bound Annexin V-PE but excluded 7-AAD was determined to be in the early stages of apoptosis. Late apoptotic/necrotic cells were quantified by dual Annexin V-PE and 7-AAD staining.
Renal cancer cell line 786-O was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI 1640 supplemented with 1 % penicillin/streptomycin and 10 % fetal bovine serum (FBS) in 5 % CO2, 37 °C cell culture incubator. The cells were harvested during the exponential phase of growth for use in the following experiments. All experiments were repeated in triplicate. Small interfering RNA mediated gene silencing of EZH2 Cells were plated in 6-well plates at a density of 0.5 9 105 cells/ml and cultured for 24 h before harvesting for experimentation. Expression of human EZH2 was knocked down with small interfering RNA (siRNA) duplexes specifically targeted to human EZH2 mRNA. Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) was used in transfection according to the protocol. The target sequences for EZH2 were: 50 -GGACTCAGAA GGCAGTGGAG-30 , 50 -CTTGAGCTGTCTCAGTCGCA30 . Nontargeting siRNA pool (D-001206-13-05; Dharmacon, Fisher Scientific, Pittsburgh, PA, USA) was used as a negative control. Cells were transfected with 1 lg of siRNA in reduced serum medium (OPTI-MEM-I; Invitrogen, Carlsbad, CA, USA) according to the protocol at 30–50 % confluence and harvested 72 h post-transfection. The RNA and protein were extracted and analyzed. Enhanced green fluorescent protein (EGFP)/EZH2 mediated overexpression of EZH2 Constructs of recombinant EGFP/EZH2 and EGFP-N1 plasmid were obtained from Thermo Scientific (Beijing, China). Transient transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, 24 h before transfection, 786-O cells were plated at 0.5–2 9 105 density, in 24-well plates. Plasmids and Lipofectamine 2000 were each diluted separately in 50 ll of serum-free Opti-MEM (Gibco BRL, Invitrogen, Carlsbad, CA, USA) and incubated for 5 min at room temperature. The two solutions were mixed at 0.8 lg plasmid to 2 ll Lipofectamine 2000 and incubated for 20 min at room temperature.
Assessment of apoptosis
Hoechst 33258 staining Apoptotic cells were examined using Hoechst 33258 staining kit (Beyotime, Haimen, China). In brief, cultured 786-O cells were seeded on cover slips in 24-well plates. Forty-eight hours after transfection, cells were fixed with 4 % paraformaldehyde at room temperature for 5 min, permeabilized with 0.2 % Triton X-100 solution in PBS for 5 min, rinsed with PBS, blocked with 5 % bovine serum albumin solution in PBS at room temperature for 2 h, and rinsed with PBS three times. Fixed cells were stained with 0.5 ml Hoechst 33258 solution in the dark for 5 min, rinsed in PBS and coverslipped with aqueous mounting medium (CTS011, BD Bioscience, San Jose, CA, USA) before observation using an Axio Imager Z2 microscope with appropriate fluorescence filters (Carl Ziess AG, Jena, Germany). Cell cycle analysis using flow cytometry Cells were harvested and single-cell suspensions containing 1 9 106 cells were fixed with 70 % alcohol. The cell cycle was examined using propidium iodide (Sigma, St. Louis, MO, USA) staining for nuclei. The fluorescence of DNA-bound propidium iodide in cells was measured with a BD FACSCalibur flow cytometer (BD Biosciences, San
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Jose, CA, USA). The results were analyzed with ModFit 3.0 software (Verity Software House, Topsham, ME, USA). Immunofluorescent microscopy Immunofluorescence staining of EZH2 and VEGF was carried out on EZH2 silencing or overexpressed 786-O cells. Briefly, cells were labeled with EZH2 (1:100) and VEGF antibody (1:200), followed by Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000, Invitrogen, Cambridge, MA, USA). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) and coverslipped with aqueous mounting medium. Slides were examined and images taken using an Axio Imager Z2 microscope with appropriate fluorescence filters (Carl Ziess AG, Jena, Germany). Each batch of cells contained a positive and a negative control. Quantitative real-time reverse-transcription polymerase chain reaction analysis TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract total RNA from EZH2 silencing or overexpressed 786-O cells and 2 lg of total RNA treated with M-MLV reverse transcriptase (Promega, Madison, WI, USA) was applied to synthesize first-strand cDNA according to the manufacturer’s instructions. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed in triplicate with Power PCR SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) using the ABI PRISM 7500 FAST Real-TIME PCR System (Applied Biosystems, Carlsbad, CA, USA) with results normalized to GAPDH expression. The relative expression was calculated using the DDCT method. Primer sequences for human EZH2 were (F) 50 -CTAGGGAGTGTTCGGTGACCA-30 and (R) 50 -ATTCTGCTGTAGGGGAGACCAAG-30 , human VEGF were (F) 50 -CGGGCAGGAGGAAGGAGCCT-30 and (R) 50 -GTGATGGTGTGGTGGCGGCA-30 and GAPDH were (F) 50 -TGGCCACTTCCGGGGTACTGT-30 and (R) 50 -CAGGTGAGCCCCGGCCTTCT-30 . Western blot analysis Cell lysates from 786-O cells were used to demonstrate changes in protein levels of EZH2 and VEGF upon EZH2 knocking down or overexpression. Lysates from each experimental group were separated in parallel on two 10 % denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5 % non-fat milk in 0.1 % tris buffered saline with
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Tween-20 (TBST), and probed using EZH2 and VEGF polyclonal antibodies. Blots were incubated with the primary antibody at 1:2,000 dilution at 4 °C overnight. After washing, the secondary antibody (horseradish peroxidaselabeled IgG anti-mouse or anti-rabbit antibody, Invitrogen, Carlsbad, CA, USA) was used at 1:3,000 dilutions for 1 h at room temperature. The Supersignal-enhanced chemoluminescence substrate (Pierce Biotechnology, Inc., Rockford, IL, USA) was applied to the probed membrane and exposed for 5 min before visualization on X-ray films (Kodak X-OMAT BT, Rochester, NY, USA). Statistical analysis Two-sided Fisher’s exact test was used to analyze correlations between expressions of EZH2 or VEGF and CCRCC patients’ clinicopathological characteristics. Spearman’s rank correlation coefficient analysis was applied to assess the correlation between the protein expressions of EZH2 and VEGF. Overall patient survival was calculated from the time of surgery to the time of death or to the time of last follow-up, at which point the data were censored. Overall survival curves were generated using the Kaplan–Meier method and the log-rank test was used to evaluate the difference between positive and negative EZH2 expression subgroups. Paired t test was used to analyze the effect of EZH2 silencing or overexpression on the expression of VEGF in 786-O renal cancer cells. SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis. A p \ 0.05 was regarded as statistically significant.
Results High expression of EZH2 and VEGF correlates with clinicopathological characteristics and poor survival in CCRCC patients The protein expression level and localization of EZH2 and VEGF were determined by immunohistochemical staining in 185 CCRCC specimens on one tissue microarray slide. EZH2 expression was negative in normal kidney tissues while positive in all tumor samples, with low expression level in 97 cases (97/185) and high expression level in 88 cases (88/185). The positive signal was in the nuclei of cancer cells (Fig. 1a, b). VEGF expression was positive in both the normal kidney and tumor tissues. In tumor tissues, VEGF was with low expression level in 82 cases (82/185) and with high expression level in 103 cases (103/185). The positive signal was in the cytoplasm of cancer cells (Fig. 1c, d). High expression level of EZH2 and VEGF correlated
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with the TNM stage (p = 0.013, p = 0.001) and distant metastases (p = 0.011, p = 0.038) of CCRCC patients (Table 2). The protein expression level of EZH2 and VEGF does not correlate with age, gender or histological grade. Spearman’s rank correlation coefficient analysis
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showed that the protein expression level of VEGF and EZH2 was positively correlated (correlation coefficient = 0.850, p \ 0.001, Fig. 1e). Kaplan–Meier survival analysis revealed that patients with positive EZH2 expression had a shorter overall survival time compared
Fig. 1 EZH2 expression was associated with poor prognosis in clear cell renal cell carcinoma patients (Immunohistochemical staining, 9400). a Low expression of EZH2 in nuclei of tumor cells (scored as 1). b High expression of EZH2 in nuclei of tumor cells (scored as 2). c Low expression of VEGF in the cytoplasm of tumor cells (scored as 1). d High expression of VEGF in the cytoplasm of tumor cells (scored as 2). e Expression of EZH2 was positively correlated with that of VEGF by Spearman’s rank coefficient correlation analysis (correlation coefficient = 0.850, p \ 0.001). f Overall survival in all CCRCC patients according to EZH2 expression by Kaplan– Meier survival analysis. p value was calculated by the log-rank test (p \ 0.001). EZH2 enhancer of zeste homolog 2, VEGF-A vascular endothelial growth factor-A, CCRCC clear cell renal cell carcinoma
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Table 2 Associations between expressions of EZH2 and VEGF and clinicopathologic characteristics in clear cell RCC patients Characteristics
EZH2 expression (N = 185) Low (N = 97) (%)
High (N = 88) (%)
B52
36 (49)
[52
p
VEGF expression (N = 185) Low (N = 82) (%)
High (N = 103) (%)
37 (51)
38 (52)
35 (48)
61 (55)
51 (45)
44 (39)
68 (61)
Male
52 (49)
53 (51)
51 (49)
54 (51)
Female
45 (56)
35 (44)
31 (39)
49 (61)
37 (48)
40 (52)
45 (42)
63 (58)
32 (33)
66 (67)
50 (58)
37 (42)
Age (years)
0.548
Gender
Histologic grade Grade 1–2 Grade 3–4
0.097
0.377
0.232
0.765 39 (51)
38 (49)
58 (54)
50 (46)
TNM stage
0.453
0.013
I–II 98
60 (61)
38 (39)
III–IV 87
37 (43)
50 (57)
Distant metastasis
0.001
0.011
0.038
Absent 127
75 (59)
52 (41)
63 (49)
64 (51)
Present 58
22 (38)
36 (62)
19 (33)
39 (67)
to patients with negative EZH2 expression (34.3 vs. 67.2, p \ 0.001, Fig. 1f). EZH2 silencing inhibits VEGF expression and cell proliferation while increases apoptosis in 786-O cells We investigated the role of EZH2 in tumor angiogenesis in renal cancer cells in vitro. SiRNA transfection was employed to knock down EZH2 expression in 786-O cells, which had high endogenous EZH2 expression. The effect of siRNA transfection on the expression of EZH2 was confirmed by immunofluorescence microscopy (Fig. 2a), RT-PCR and Western blot analysis. The amount of EZH2 mRNA or protein, normalized over GAPDH, was reduced up to 85 % compared to negative control cells (data not shown), indicating the successful knocking down of EZH2 gene in 786-O cells. In vitro silencing of EZH2 inhibited both the mRNA (Fig. 2b) and protein (Fig. 2c, d) expression of VEGF, suggesting the critical role of EZH2 in renal cancer angiogenesis. EZH2 silencing increased apoptosis of 786-O cells significantly, as shown by Hoechst staining of the tumor cell nuclei (Fig. 2e), the apoptotic index (Fig. 2f) and flow cytometry analyses (Fig. 2g). The number of cells arrested in the S and G2 phase after EZH2 silencing was decreased (Fig. 2h). Similar results were obtained in three independent experiments.
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p
EZH2 overexpression promotes VEGF expression and cell proliferation while inhibits apoptosis in 786-O cells We investigated the role of EZH2 overexpression in tumor angiogenesis in renal cancer cells in vitro. EGFP/EZH2 was employed to overexpress EZH2 expression in 786-O cells. The effect of EGFP/EZH2 transfection on the expression of EZH2 was confirmed by immunofluorescence microscopy (Fig. 3a), RT-PCR and Western blot analysis. The amount of EZH2 mRNA or protein, normalized over GAPDH, was increased up to onefold compared to negative control cells (data now shown), indicating the successful overexpression of EZH2 gene in 786-O cells. EZH2 overexpression promoted both the mRNA (Fig. 3b) and protein (Fig. 3c, d) expression of VEGF, suggesting the critical role of EZH2 in tumor angiogenesis in renal cancer cells. EZH2 overexpression inhibited apoptosis of 786-O cells significantly, as shown by Hoechst staining of the tumor cell nuclei (Fig. 3e), the apoptotic index (Fig. 3f) and flow cytometry analyses (Fig. 3g). Cell cycle analysis with flow cytometry revealed that the proportion of cells arrested in the S and G2/M phase was 68.54 % in EGFP/EZH2 overexpressing cells, compared with 47.94 % in EGFP cells and 47.29 % in negative control cells (Fig. 2g). The result suggests that EZH2 plays a critical role in cell growth and proliferation in 786-O cells in vitro. Similar results were obtained in three independent experiments.
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Fig. 2 EZH2 siRNAs inhibit VEGF and cell proliferation while increase apoptosis in 786-O cell line. a Representative images of 786-O cells transfected with EZH2_siRNA#1, #2 and NT_siRNA and stained for EZH2 (red), VEGF (green) and DAPI (blue) (immunofluorescence, scale bar 50 lm). b EZH2_siRNA#1 or #2 downregulated VEGF mRNA expression levels compared to NT_siRNA as shown in quantitative real-time RT-PCR. c EZH2_siRNA#1 or #2 downregulated VEGF protein levels compared to NT_siRNA as shown in Western blot analysis and d the relative quantification normalized by GAPDH expression. e Increased apoptosis after EZH2 silencing in 786-O cells stained with Hoechst 33258 after 72 h post-
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transfection (immunofluorescence, scale bar 50 lm). f Increased apoptotic indexes in 786-O cells transfected with EZH2_siRNA#1 or #2 compared to NT_siRNA 72 h post-transfection. g Increased apoptosis in 786-O cells transfected with EZH2_siRNA#1 or #2 compared to NT_siRNA 72 h post-transfection analyzed by flow cytometry. h Decreased S and G2/M phases in 786-O cells transfected with EZH2_siRNA#1 or #2 compared to NT_siRNA 72 h posttransfection analyzed by flow cytometry. Each bar represented the mean ± SD. Results were representative of three independent experiments, *, àp \ 0.05. EZH2 enhancer of zeste homolog 2, DAPI 4,6-diamidino-2-phenylindole, NT_siRNA non-targeting siRNA
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Fig. 3 EZH2 overexpression promotes VEGF expression and cell proliferation while inhibits apoptosis in 786-O cells. a Representative images of 786-O cells transfected with EGFP/EZH2 and EGFP-N1 (immunofluorescence, scale bar 50 lm). b EGFP/EZH2 upregulated VEGF mRNA expression level compared to EGFP-N1 and the negative control as shown in quantitative real-time RT-PCR. c EGFP/ EZH2 upregulated VEGF protein level compared to EGFP-N1 and the negative control as shown in Western blot analysis and d the relative quantification normalized by GAPDH expression. e Decreased apoptosis after EZH2 overexpression in 786-O cells stained with Hoechst 33258 after 72 h post-transfection (immunofluorescence, scale bar
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50 lm). f Decreased apoptotic indexes in 786-O cells transfected with EGFP/EZH2 compared with EGFP-N1 and the negative control 72 h post-transfection. g Decreased apoptosis in 786-O cells transfected with EGFP/EZH2 compared with EGFP-N1 and the negative control 72 h post-transfection analyzed by flow cytometry. h Increased S and G2/M phases in 786-O cells transfected with EGFP/EZH2 compared with EGFP-N1 and the negative control 72 h post-transfection analyzed by flow cytometry. Each bar represented the mean ± SD. Results were representative of three independent experiments, *, àp \ 0.05. EGFP enhanced green fluorescent protein, EZH2 enhancer of zeste homolog 2
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Discussion In the present study, we investigated the roles and functions of EZH2 on angiogenesis in clear cell renal cell carcinoma. EZH2 expression level was increased with the development of renal cancer with negative expression in normal renal tissues, low expression in highly differentiated renal cancers and high expression in poorly differentiated renal cancers. We employed tissue microarray technology and detected, respectively, the expression level of EZH2 and VEGF in CCRCC tissues using immunohistochemistry. The present study has shown that EZH2 expression level increased with the progression of CCRCC, suggesting EZH2 as a potential clinical biomarker for renal cancer. Compared with early staged tumors, the late staged invasive renal cancer had significant detectable EZH2 and VEGF expressions. The expression of EZH2 and VEGF was more frequent in high-grade renal tumors than in lowgrade tumors. The expression level of EZH2 and VEGF correlated with TNM stage and distant metastasis of renal cancer. The expression level of EZH2 correlated positively with that of VEGF in CCRCC. Other studies have shown that EZH2 plays an important role for the growth control of colon cancer through arresting colon carcinoma cells at the G1/S transition [18, 19]. EZH2 overexpression is associated with the aggressive behavior of prostate and breast cancer [20, 21]. The present study suggests that EZH2 overexpression in the progression and development of renal cancer may lead to increased angiogenesis which promotes renal carcinogenesis. We examined the role of EZH2 in renal cancer by investigating the effect of EZH2 silencing or overexpression on VEGF expression, cell growth and apoptosis in renal cancer cell line 786-O. Our results showed that silencing EZH2 in 786-O cells inhibited the expression of VEGF, while overexpressing EZH2 had the opposite effect. Silencing EZH2 suppressed cell growth and induced G1 arrest and apoptosis, while overexpressing EZH2 promoted cell growth and inhibited apoptosis in vitro. These results are in agreement with the investigations in gastric, lung, and pancreatic cancers, in which knocking down EZH2 inhibited cell proliferation and promoted apoptosis [13, 14, 22]. In conclusion, the present study demonstrates the relationship between the expression of EZH2 and VEGF in the progression and development of CCRCC. EZH2 expression accelerates cell cycle and antiapoptosis in CCRCC. These results suggest EZH2 targeting might be an attractive therapeutic approach in the treatment of renal cancer.
49 Acknowledgments This study was supported by Grants from the National Natural Science Foundation of China for Young Investigators (No. 81102237) and Jilin Provincial Natural Science Foundation (Nos. 201215047 and 201115057). Conflict of interest
There are no conflicts of interest.
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RESEARCH ARTICLE
EZH2 promotes tumor progression by increasing VEGF expression in clear cell renal cell carcinoma Z. Q. Xu • L. Zhang • B. S. Gao • Y. G. Wan X. H. Zhang • B. Chen • Y. T. Wang • N. Sun • Y. W. Fu
•
Received: 31 March 2014 / Accepted: 11 June 2014 / Published online: 2 July 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Objectives The present study is to evaluate the expression level of enhancer of zeste homolog 2 (EZH2) and vascular endothelial growth factor (VEGF), and analyze their correlations with clinicopathological characteristics and survival in patients with clear cell renal cell carcinoma (CCRCC). The effect of EZH2 on apoptosis and cell proliferation in 786-O renal cancer cell line is investigated. Methods The expression level of EZH2 and VEGF was detected in 185 primary CCRCC patients’ tissues using tissue microarray and immunohistochemistry. Small interfering RNA or enhanced green fluorescent protein transfection was employed to investigate the effect of EZH2 inhibition or overexpression on VEGF expression, apoptosis and cell proliferation in 786-O cells using flow cytometry, immunofluorescence microscopy, quantitative real-time reverse-transcription polymerase chain reaction and Western blot analysis. Results High expression level of EZH2 and VEGF was observed in advanced CCRCC and correlated with the TNM stage (p = 0.013, p = 0.001) and distant metastasis
Z. Q. Xu B. S. Gao Y. T. Wang N. Sun Y. W. Fu (&) Urology Center, The First Hospital, Jilin University, Changchun 130021, Jilin, People’s Republic of China e-mail: [email protected] Z. Q. Xu e-mail: [email protected] Z. Q. Xu Y. G. Wan X. H. Zhang B. Chen Department of Urology, Tongliao Hospital, Tongliao 028000, Inner Mongolia, People’s Republic of China L. Zhang Department of Pharmacy, Tongliao Hospital, Tongliao 028000, Inner Mongolia, People’s Republic of China
(p = 0.011, p = 0.038), respectively. EZH2 was positively correlated with VEGF in CCRCC tissues (correlation coefficient = 0.850, p \ 0.001). Kaplan–Meier survival analysis revealed that patients with positive EZH2 expression had a shorter overall survival time compared to patients with negative EZH2 expression (34.3 vs. 67.2, p \ 0.001). In 786-O cells, EZH2 silencing inhibited VEGF expression and cell proliferation while increasing apoptosis (p \ 0.001). EZH2 overexpression promoted VEGF expression and cell proliferation while inhibiting apoptosis (p \ 0.001). Conclusions EZH2 correlates positively with VEGF and associates with adverse clinicopathologic characteristics and shorter survival time in CCRCC patients. EZH2 accelerates antiapoptosis and cell cycle in 786-O cells. Keywords EZH2 VEGF Apoptosis Proliferation Clear cell renal cell carcinoma
Introduction Renal cell carcinoma (RCC), comprising 90 % of primary malignant renal tumors in adults, originates from the renal parenchyma and remains a major cause of morbidity and mortality [1]. Clear cell renal cell carcinoma (CCRCC) is the major histological subtype of RCC, comprising around 71 % of all RCC cases [2]. Approximately, 40 % of CCRCC patients eventually die of cancer progression with the highest fatality rate among the common urologic malignancies. The enhancer of zeste homolog 2 (EZH2), located on chromosome 7q35, is a member of the polycomb-group (PcG) genes required for the stable transmission of gene expression patterns to progeny cells throughout
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development. It functions as a transcriptional regulator for the maintenance of cell cycle regulation, cell identity and oncogenesis. EZH2, embryonic ectoderm development (EED), Yin-Yang-1 (YY1) and suppressor of zeste 12 (SUZ12) constitute the polycomb repressive complex (PRC) 2 [3]. EZH2 serves as a histone methyltransferase and results in silencing of various genes, including tumor suppressor genes, through direct methylation of lysine-27 of histone H3. Dysregulated expression of EZH2 is associated with human hematologic malignancies, such as malignant myeloid disorders [4] and lymphomas [5, 6] as well as epithelial malignancies including prostate [7], bladder [8], and skin cancers [9]. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes angiogenesis in tumorigenesis [10]. VEGF has been implicated with tumor growth, metastasis and poor prognosis in renal cancer and anti-VEGF therapy has been widely used for the treatment of RCC [11]. Studies have shown that VEGF-mediated down-regulation of miR-101 results in increased expression of EZH2 in angiogenic endothelial cells and decreased expression of EZH2 inhibits vascularization in glioblastomas. These results suggest a possible therapeutic potential for EZH2 inhibition in tumors with aberrant vascularization [12]. EZH2 inhibition increases the apoptotic activity and accelerates the cell cycle in small cell lung carcinoma [13] and in patients with cholangiocarcinoma [14]. Although both EZH2 and VEGF have been correlated with poor prognosis of RCC, little is known about the role EZH2 plays in tumor angiogenesis and the effect of EZH2 on apoptosis and cell cycle regulation in CCRCC. The objective of this study is to determine the expressions of EZH2 and VEGF and their correlations with clinicopathological characteristics and survival in patients with CCRCC. We also explored the effect of EZH2 on apoptosis and cell proliferation in 786-O renal cancer cell line.
Materials and methods Patients’ parameters One hundred and eighty-five renal cancer patients treated in the Urology Center of the First Hospital, Jilin University in Changchun China from 2000 to 2013 were selected for this study. Written informed consents were obtained from all patients according to the rules of Jilin University Ethnics Committee. The inclusion criteria were: CCRCC and no radiotherapy or chemotherapy. The clinicopathological characteristics, defined by the World Health Organization (WHO) criteria and the seventh edition of the American Joint Committee of Cancer (AJCC) staging system [2, 15], included age, gender, histologic grade, TNM stage and
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distant metastasis (Table 1). Tissue specimens from these patients were collected from the Department of Pathology and the Urology Center in the First Hospital of Jilin University including 185 paraffin-embedded primary tumor samples. Normal kidney tissues, 5 cm to the tumor area were used as normal controls. The overall survival time of the patients after tumor removal was 58.5 months (range 16–98 months). At the time of censoring the data, the follow-up information of 48 (26 %) patients was lost due to death or other reasons. Tissue microarray and immunohistochemical staining Tissue microarray construction and immunohistochemical staining were performed as described previously [16, 17]. Briefly, tissue microarray slides were stained with antibodies to EZH2 (mouse anti-human polyclonal antibody, Cell Signaling Technology, Inc., Danvers, MA, USA) and VEGF (rabbit anti-human polyclonal antibody, Abcam, Cambridge, MA, USA) based on the standard streptavidin– biotin complex method. Prostate adenocarcinoma tissues were used as positive controls. Phosphate-buffered saline (PBS, pH = 7.4) replaced the primary antibody as a negative control. The samples were analyzed under a 409 objective lens of an Axio Imager Z2 microscope (Carl Ziess AG, Jena, Germany). Ten fields were randomly selected within three randomly chosen representative tissue cores of a specimen on the tissue microarray slide. One hundred tumor cells per patient were analyzed. Cells with dark brown particles in the nuclei or cytoplasm were regarded as having positive protein expression. The immunohistochemical staining was Table 1 patients
Clinical pathologic characteristics of clear cell RCC
Characteristics
No. of patients (N = 185) (%)
Age (year) B52
73
[52
112
Gender Male Female
105 80
Histologic grade Grade 1–2
77
Grade 3–4
108
TNM stage I–II
98
III–IV
87
Distant metastasis Absent
127
Present
58
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recorded as: no expression (absent staining = 0), low expression (C30 % of tumor cells with weak staining intensity = 1), or high expression (C30 % of tumor cells with strong staining intensity = 2). No expression was regarded as negative and low or high expression as positive.
A total of 100 ll of the mixture was added to 400 ll of culture medium in each well. Cells were incubated at 37 °C in a 5 % CO2 incubator for 72 h and then tested for transgene expression.
Cell line and culture
Apoptosis in EZH2 silencing or overexpressed 786-O cells was quantified using flow cytometry. Cells were harvested and pooled after 72 h transfection and stained with Annexin V-PE (BD Biosciences, San Jose, CA, USA), a marker of early phase apoptosis, and with 7-aminoactinomycin (7AAD) (BD Biosciences, San Jose, CA, USA), an indicator of late apoptosis. Briefly, cells were rinsed twice in PBS and suspended in binding buffer containing 10 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/ NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Staining mixture of 5 ll Annexin V-PE and 5 ll 7-AAD was added to 100 ll of cell suspension (1 9 106 cells/ml). Cells were incubated in the dark for 15 min at room temperature and then analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Percentage of apoptotic cells that bound Annexin V-PE but excluded 7-AAD was determined to be in the early stages of apoptosis. Late apoptotic/necrotic cells were quantified by dual Annexin V-PE and 7-AAD staining.
Renal cancer cell line 786-O was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI 1640 supplemented with 1 % penicillin/streptomycin and 10 % fetal bovine serum (FBS) in 5 % CO2, 37 °C cell culture incubator. The cells were harvested during the exponential phase of growth for use in the following experiments. All experiments were repeated in triplicate. Small interfering RNA mediated gene silencing of EZH2 Cells were plated in 6-well plates at a density of 0.5 9 105 cells/ml and cultured for 24 h before harvesting for experimentation. Expression of human EZH2 was knocked down with small interfering RNA (siRNA) duplexes specifically targeted to human EZH2 mRNA. Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) was used in transfection according to the protocol. The target sequences for EZH2 were: 50 -GGACTCAGAA GGCAGTGGAG-30 , 50 -CTTGAGCTGTCTCAGTCGCA30 . Nontargeting siRNA pool (D-001206-13-05; Dharmacon, Fisher Scientific, Pittsburgh, PA, USA) was used as a negative control. Cells were transfected with 1 lg of siRNA in reduced serum medium (OPTI-MEM-I; Invitrogen, Carlsbad, CA, USA) according to the protocol at 30–50 % confluence and harvested 72 h post-transfection. The RNA and protein were extracted and analyzed. Enhanced green fluorescent protein (EGFP)/EZH2 mediated overexpression of EZH2 Constructs of recombinant EGFP/EZH2 and EGFP-N1 plasmid were obtained from Thermo Scientific (Beijing, China). Transient transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, 24 h before transfection, 786-O cells were plated at 0.5–2 9 105 density, in 24-well plates. Plasmids and Lipofectamine 2000 were each diluted separately in 50 ll of serum-free Opti-MEM (Gibco BRL, Invitrogen, Carlsbad, CA, USA) and incubated for 5 min at room temperature. The two solutions were mixed at 0.8 lg plasmid to 2 ll Lipofectamine 2000 and incubated for 20 min at room temperature.
Assessment of apoptosis
Hoechst 33258 staining Apoptotic cells were examined using Hoechst 33258 staining kit (Beyotime, Haimen, China). In brief, cultured 786-O cells were seeded on cover slips in 24-well plates. Forty-eight hours after transfection, cells were fixed with 4 % paraformaldehyde at room temperature for 5 min, permeabilized with 0.2 % Triton X-100 solution in PBS for 5 min, rinsed with PBS, blocked with 5 % bovine serum albumin solution in PBS at room temperature for 2 h, and rinsed with PBS three times. Fixed cells were stained with 0.5 ml Hoechst 33258 solution in the dark for 5 min, rinsed in PBS and coverslipped with aqueous mounting medium (CTS011, BD Bioscience, San Jose, CA, USA) before observation using an Axio Imager Z2 microscope with appropriate fluorescence filters (Carl Ziess AG, Jena, Germany). Cell cycle analysis using flow cytometry Cells were harvested and single-cell suspensions containing 1 9 106 cells were fixed with 70 % alcohol. The cell cycle was examined using propidium iodide (Sigma, St. Louis, MO, USA) staining for nuclei. The fluorescence of DNA-bound propidium iodide in cells was measured with a BD FACSCalibur flow cytometer (BD Biosciences, San
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Jose, CA, USA). The results were analyzed with ModFit 3.0 software (Verity Software House, Topsham, ME, USA). Immunofluorescent microscopy Immunofluorescence staining of EZH2 and VEGF was carried out on EZH2 silencing or overexpressed 786-O cells. Briefly, cells were labeled with EZH2 (1:100) and VEGF antibody (1:200), followed by Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000, Invitrogen, Cambridge, MA, USA). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA) and coverslipped with aqueous mounting medium. Slides were examined and images taken using an Axio Imager Z2 microscope with appropriate fluorescence filters (Carl Ziess AG, Jena, Germany). Each batch of cells contained a positive and a negative control. Quantitative real-time reverse-transcription polymerase chain reaction analysis TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract total RNA from EZH2 silencing or overexpressed 786-O cells and 2 lg of total RNA treated with M-MLV reverse transcriptase (Promega, Madison, WI, USA) was applied to synthesize first-strand cDNA according to the manufacturer’s instructions. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) analysis was performed in triplicate with Power PCR SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) using the ABI PRISM 7500 FAST Real-TIME PCR System (Applied Biosystems, Carlsbad, CA, USA) with results normalized to GAPDH expression. The relative expression was calculated using the DDCT method. Primer sequences for human EZH2 were (F) 50 -CTAGGGAGTGTTCGGTGACCA-30 and (R) 50 -ATTCTGCTGTAGGGGAGACCAAG-30 , human VEGF were (F) 50 -CGGGCAGGAGGAAGGAGCCT-30 and (R) 50 -GTGATGGTGTGGTGGCGGCA-30 and GAPDH were (F) 50 -TGGCCACTTCCGGGGTACTGT-30 and (R) 50 -CAGGTGAGCCCCGGCCTTCT-30 . Western blot analysis Cell lysates from 786-O cells were used to demonstrate changes in protein levels of EZH2 and VEGF upon EZH2 knocking down or overexpression. Lysates from each experimental group were separated in parallel on two 10 % denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto nitrocellulose membranes, blocked with 5 % non-fat milk in 0.1 % tris buffered saline with
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Tween-20 (TBST), and probed using EZH2 and VEGF polyclonal antibodies. Blots were incubated with the primary antibody at 1:2,000 dilution at 4 °C overnight. After washing, the secondary antibody (horseradish peroxidaselabeled IgG anti-mouse or anti-rabbit antibody, Invitrogen, Carlsbad, CA, USA) was used at 1:3,000 dilutions for 1 h at room temperature. The Supersignal-enhanced chemoluminescence substrate (Pierce Biotechnology, Inc., Rockford, IL, USA) was applied to the probed membrane and exposed for 5 min before visualization on X-ray films (Kodak X-OMAT BT, Rochester, NY, USA). Statistical analysis Two-sided Fisher’s exact test was used to analyze correlations between expressions of EZH2 or VEGF and CCRCC patients’ clinicopathological characteristics. Spearman’s rank correlation coefficient analysis was applied to assess the correlation between the protein expressions of EZH2 and VEGF. Overall patient survival was calculated from the time of surgery to the time of death or to the time of last follow-up, at which point the data were censored. Overall survival curves were generated using the Kaplan–Meier method and the log-rank test was used to evaluate the difference between positive and negative EZH2 expression subgroups. Paired t test was used to analyze the effect of EZH2 silencing or overexpression on the expression of VEGF in 786-O renal cancer cells. SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis. A p \ 0.05 was regarded as statistically significant.
Results High expression of EZH2 and VEGF correlates with clinicopathological characteristics and poor survival in CCRCC patients The protein expression level and localization of EZH2 and VEGF were determined by immunohistochemical staining in 185 CCRCC specimens on one tissue microarray slide. EZH2 expression was negative in normal kidney tissues while positive in all tumor samples, with low expression level in 97 cases (97/185) and high expression level in 88 cases (88/185). The positive signal was in the nuclei of cancer cells (Fig. 1a, b). VEGF expression was positive in both the normal kidney and tumor tissues. In tumor tissues, VEGF was with low expression level in 82 cases (82/185) and with high expression level in 103 cases (103/185). The positive signal was in the cytoplasm of cancer cells (Fig. 1c, d). High expression level of EZH2 and VEGF correlated
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with the TNM stage (p = 0.013, p = 0.001) and distant metastases (p = 0.011, p = 0.038) of CCRCC patients (Table 2). The protein expression level of EZH2 and VEGF does not correlate with age, gender or histological grade. Spearman’s rank correlation coefficient analysis
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showed that the protein expression level of VEGF and EZH2 was positively correlated (correlation coefficient = 0.850, p \ 0.001, Fig. 1e). Kaplan–Meier survival analysis revealed that patients with positive EZH2 expression had a shorter overall survival time compared
Fig. 1 EZH2 expression was associated with poor prognosis in clear cell renal cell carcinoma patients (Immunohistochemical staining, 9400). a Low expression of EZH2 in nuclei of tumor cells (scored as 1). b High expression of EZH2 in nuclei of tumor cells (scored as 2). c Low expression of VEGF in the cytoplasm of tumor cells (scored as 1). d High expression of VEGF in the cytoplasm of tumor cells (scored as 2). e Expression of EZH2 was positively correlated with that of VEGF by Spearman’s rank coefficient correlation analysis (correlation coefficient = 0.850, p \ 0.001). f Overall survival in all CCRCC patients according to EZH2 expression by Kaplan– Meier survival analysis. p value was calculated by the log-rank test (p \ 0.001). EZH2 enhancer of zeste homolog 2, VEGF-A vascular endothelial growth factor-A, CCRCC clear cell renal cell carcinoma
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Table 2 Associations between expressions of EZH2 and VEGF and clinicopathologic characteristics in clear cell RCC patients Characteristics
EZH2 expression (N = 185) Low (N = 97) (%)
High (N = 88) (%)
B52
36 (49)
[52
p
VEGF expression (N = 185) Low (N = 82) (%)
High (N = 103) (%)
37 (51)
38 (52)
35 (48)
61 (55)
51 (45)
44 (39)
68 (61)
Male
52 (49)
53 (51)
51 (49)
54 (51)
Female
45 (56)
35 (44)
31 (39)
49 (61)
37 (48)
40 (52)
45 (42)
63 (58)
32 (33)
66 (67)
50 (58)
37 (42)
Age (years)
0.548
Gender
Histologic grade Grade 1–2 Grade 3–4
0.097
0.377
0.232
0.765 39 (51)
38 (49)
58 (54)
50 (46)
TNM stage
0.453
0.013
I–II 98
60 (61)
38 (39)
III–IV 87
37 (43)
50 (57)
Distant metastasis
0.001
0.011
0.038
Absent 127
75 (59)
52 (41)
63 (49)
64 (51)
Present 58
22 (38)
36 (62)
19 (33)
39 (67)
to patients with negative EZH2 expression (34.3 vs. 67.2, p \ 0.001, Fig. 1f). EZH2 silencing inhibits VEGF expression and cell proliferation while increases apoptosis in 786-O cells We investigated the role of EZH2 in tumor angiogenesis in renal cancer cells in vitro. SiRNA transfection was employed to knock down EZH2 expression in 786-O cells, which had high endogenous EZH2 expression. The effect of siRNA transfection on the expression of EZH2 was confirmed by immunofluorescence microscopy (Fig. 2a), RT-PCR and Western blot analysis. The amount of EZH2 mRNA or protein, normalized over GAPDH, was reduced up to 85 % compared to negative control cells (data not shown), indicating the successful knocking down of EZH2 gene in 786-O cells. In vitro silencing of EZH2 inhibited both the mRNA (Fig. 2b) and protein (Fig. 2c, d) expression of VEGF, suggesting the critical role of EZH2 in renal cancer angiogenesis. EZH2 silencing increased apoptosis of 786-O cells significantly, as shown by Hoechst staining of the tumor cell nuclei (Fig. 2e), the apoptotic index (Fig. 2f) and flow cytometry analyses (Fig. 2g). The number of cells arrested in the S and G2 phase after EZH2 silencing was decreased (Fig. 2h). Similar results were obtained in three independent experiments.
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p
EZH2 overexpression promotes VEGF expression and cell proliferation while inhibits apoptosis in 786-O cells We investigated the role of EZH2 overexpression in tumor angiogenesis in renal cancer cells in vitro. EGFP/EZH2 was employed to overexpress EZH2 expression in 786-O cells. The effect of EGFP/EZH2 transfection on the expression of EZH2 was confirmed by immunofluorescence microscopy (Fig. 3a), RT-PCR and Western blot analysis. The amount of EZH2 mRNA or protein, normalized over GAPDH, was increased up to onefold compared to negative control cells (data now shown), indicating the successful overexpression of EZH2 gene in 786-O cells. EZH2 overexpression promoted both the mRNA (Fig. 3b) and protein (Fig. 3c, d) expression of VEGF, suggesting the critical role of EZH2 in tumor angiogenesis in renal cancer cells. EZH2 overexpression inhibited apoptosis of 786-O cells significantly, as shown by Hoechst staining of the tumor cell nuclei (Fig. 3e), the apoptotic index (Fig. 3f) and flow cytometry analyses (Fig. 3g). Cell cycle analysis with flow cytometry revealed that the proportion of cells arrested in the S and G2/M phase was 68.54 % in EGFP/EZH2 overexpressing cells, compared with 47.94 % in EGFP cells and 47.29 % in negative control cells (Fig. 2g). The result suggests that EZH2 plays a critical role in cell growth and proliferation in 786-O cells in vitro. Similar results were obtained in three independent experiments.
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Fig. 2 EZH2 siRNAs inhibit VEGF and cell proliferation while increase apoptosis in 786-O cell line. a Representative images of 786-O cells transfected with EZH2_siRNA#1, #2 and NT_siRNA and stained for EZH2 (red), VEGF (green) and DAPI (blue) (immunofluorescence, scale bar 50 lm). b EZH2_siRNA#1 or #2 downregulated VEGF mRNA expression levels compared to NT_siRNA as shown in quantitative real-time RT-PCR. c EZH2_siRNA#1 or #2 downregulated VEGF protein levels compared to NT_siRNA as shown in Western blot analysis and d the relative quantification normalized by GAPDH expression. e Increased apoptosis after EZH2 silencing in 786-O cells stained with Hoechst 33258 after 72 h post-
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transfection (immunofluorescence, scale bar 50 lm). f Increased apoptotic indexes in 786-O cells transfected with EZH2_siRNA#1 or #2 compared to NT_siRNA 72 h post-transfection. g Increased apoptosis in 786-O cells transfected with EZH2_siRNA#1 or #2 compared to NT_siRNA 72 h post-transfection analyzed by flow cytometry. h Decreased S and G2/M phases in 786-O cells transfected with EZH2_siRNA#1 or #2 compared to NT_siRNA 72 h posttransfection analyzed by flow cytometry. Each bar represented the mean ± SD. Results were representative of three independent experiments, *, àp \ 0.05. EZH2 enhancer of zeste homolog 2, DAPI 4,6-diamidino-2-phenylindole, NT_siRNA non-targeting siRNA
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Fig. 3 EZH2 overexpression promotes VEGF expression and cell proliferation while inhibits apoptosis in 786-O cells. a Representative images of 786-O cells transfected with EGFP/EZH2 and EGFP-N1 (immunofluorescence, scale bar 50 lm). b EGFP/EZH2 upregulated VEGF mRNA expression level compared to EGFP-N1 and the negative control as shown in quantitative real-time RT-PCR. c EGFP/ EZH2 upregulated VEGF protein level compared to EGFP-N1 and the negative control as shown in Western blot analysis and d the relative quantification normalized by GAPDH expression. e Decreased apoptosis after EZH2 overexpression in 786-O cells stained with Hoechst 33258 after 72 h post-transfection (immunofluorescence, scale bar
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50 lm). f Decreased apoptotic indexes in 786-O cells transfected with EGFP/EZH2 compared with EGFP-N1 and the negative control 72 h post-transfection. g Decreased apoptosis in 786-O cells transfected with EGFP/EZH2 compared with EGFP-N1 and the negative control 72 h post-transfection analyzed by flow cytometry. h Increased S and G2/M phases in 786-O cells transfected with EGFP/EZH2 compared with EGFP-N1 and the negative control 72 h post-transfection analyzed by flow cytometry. Each bar represented the mean ± SD. Results were representative of three independent experiments, *, àp \ 0.05. EGFP enhanced green fluorescent protein, EZH2 enhancer of zeste homolog 2
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Discussion In the present study, we investigated the roles and functions of EZH2 on angiogenesis in clear cell renal cell carcinoma. EZH2 expression level was increased with the development of renal cancer with negative expression in normal renal tissues, low expression in highly differentiated renal cancers and high expression in poorly differentiated renal cancers. We employed tissue microarray technology and detected, respectively, the expression level of EZH2 and VEGF in CCRCC tissues using immunohistochemistry. The present study has shown that EZH2 expression level increased with the progression of CCRCC, suggesting EZH2 as a potential clinical biomarker for renal cancer. Compared with early staged tumors, the late staged invasive renal cancer had significant detectable EZH2 and VEGF expressions. The expression of EZH2 and VEGF was more frequent in high-grade renal tumors than in lowgrade tumors. The expression level of EZH2 and VEGF correlated with TNM stage and distant metastasis of renal cancer. The expression level of EZH2 correlated positively with that of VEGF in CCRCC. Other studies have shown that EZH2 plays an important role for the growth control of colon cancer through arresting colon carcinoma cells at the G1/S transition [18, 19]. EZH2 overexpression is associated with the aggressive behavior of prostate and breast cancer [20, 21]. The present study suggests that EZH2 overexpression in the progression and development of renal cancer may lead to increased angiogenesis which promotes renal carcinogenesis. We examined the role of EZH2 in renal cancer by investigating the effect of EZH2 silencing or overexpression on VEGF expression, cell growth and apoptosis in renal cancer cell line 786-O. Our results showed that silencing EZH2 in 786-O cells inhibited the expression of VEGF, while overexpressing EZH2 had the opposite effect. Silencing EZH2 suppressed cell growth and induced G1 arrest and apoptosis, while overexpressing EZH2 promoted cell growth and inhibited apoptosis in vitro. These results are in agreement with the investigations in gastric, lung, and pancreatic cancers, in which knocking down EZH2 inhibited cell proliferation and promoted apoptosis [13, 14, 22]. In conclusion, the present study demonstrates the relationship between the expression of EZH2 and VEGF in the progression and development of CCRCC. EZH2 expression accelerates cell cycle and antiapoptosis in CCRCC. These results suggest EZH2 targeting might be an attractive therapeutic approach in the treatment of renal cancer.
49 Acknowledgments This study was supported by Grants from the National Natural Science Foundation of China for Young Investigators (No. 81102237) and Jilin Provincial Natural Science Foundation (Nos. 201215047 and 201115057). Conflict of interest
There are no conflicts of interest.
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