Journal: Nature Communications

Article Title: Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

doi: 10.1038/s41467-022-32311-2

Figure Lengend Snippet: a Volcano plot of differentially expressed circRNAs in 12 paired human GBM samples. b Box plot of the length distribution of circORFs in tumour-upregulated circRNAs and the corresponding mORFs of the source genes. Data are presented as boxes containing the median (centre line), the first and third quartiles (box limits). The whiskers indicate the maxima and minima. c Expression distribution (x-axis) and length distribution (y-axis) of circORFs in tumour-upregulated circRNAs. The red dots represent the 10 candidate circRNAs. d Heatmap of the fold change values of the 10 circRNAs in ( c ). e Pathways negatively correlated with circEZH2 in GSEA. f Correlation analysis between circEZH2 and the functional NK cell-associated marker gene set (27 genes) in GSEA. The heatmap on the right shows the Pearson correlation coefficient between each marker and circEZH2. The vertical bars in the figure indicate the ranks and enrichment scores of the 27 genes as determined by GSEA. Ten core enriched genes are marked in the plot. g Illustration of the annotated genomic region of EZH2, the putative different RNA splicing forms. Convergent and divergent primers were designed to amplify the linear- or back-spliced products. h Left, PCR analysis using the indicated primers. Right, subsequent Sanger sequencing identified the junction sequence of circEZH2 in MES28 cells. i Left, illustration of the circEZH2 junction-specific FISH probe and circEZH2 shRNA target site. Right, FISH was performed to identify the subcellular localization of circEZH2. CircEZH2 OV plasmids and shRNA were used independently or in combination to verify the specificity of these probes. Scale bar, 10 μm. j Relative circEZH2 RNA levels in NHAs and GSC lines. 456 vs NHA, P = 0.0389; 4121 vs NHA, P = 0.0056; MES28 vs NHA, P = 0.0056; GSC23 vs NHA, P = 0.0005. k Fold change in circEZH2 expression in tumour specimens and paired adjacent brain tissues in a cohort of high-grade glioma patients ( n = 63). l Relative circEZH2 expression levels in the same cohort ( n = 63). Two-sided paired t test, P = 0.0013. The data in ( h )–( l ) are pooled from three independent experiments. The data are presented as the mean ± SD. Unpaired two-tailed Student’s t test was used to determine the significance of differences between the indicated groups where applicable. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: A rabbit polyclonal antibody specific for EZH2-92aa (1:500) was produced by GenScript Biotech (Jiangsu, China).

Techniques: Expressing, Functional Assay, Marker, Sequencing, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

doi: 10.1038/s41467-022-32311-2

Figure Lengend Snippet: a Illustration of the circEZH2-encoded peptide EZH2-92aa. The unique C-terminus of EZH2-92aa (in red) is produced by an ORF spanning more than 360° in circEZH2. A custom antibody against the indicated unique C-terminal sequence was produced. b 293T cells transfected with the circEZH2 plasmid were subjected to polysome profiling. CircEZH2 was detected by qPCR in the indicated fractions. CircHIPK3 and EZH2 served as the negative and positive controls, respectively. c Illustration of endogenous circEZH2, the del-ATG circEZH2 construct and the linearized EZH2-92aa-3×Flag construct. d Immunoblot of cells overexpressing the above constructs using the custom anti-EZH2-92aa and anti-flag antibodies. VA, vector alone. e Identification of the unique C-terminal peptide sequence of EZH2-92aa in 293T cells overexpressing circEZH2 and in MES28 GSCs by MS. f Top, EZH2-92aa expression levels were measured in NHAs and several established GSC lines. Bottom, immunoblot of EZH2-92aa in seven randomly selected paired GBM samples using the custom anti-EZH2-92aa antibody. g Semiquantitative analysis of the EZH2-92aa expression level based on greyscale analysis in the aforementioned cohort of 63 high-grade glioma samples. Two-sided paired t test, P = 3.14e−05. h Survival analysis of patients stratified by EZH2-92aa expression (with the median expression score as the cut-off value) in the same cohort. Log-rank test, P = 0.0066. The data in ( b ), ( d ), ( e ) and ( f ) are pooled from three independent experiments and are presented as the mean ± SD. Source data are provided as a Source data file.

Article Snippet: A rabbit polyclonal antibody specific for EZH2-92aa (1:500) was produced by GenScript Biotech (Jiangsu, China).

Techniques: Produced, Sequencing, Transfection, Plasmid Preparation, Construct, Western Blot, Expressing

Journal: Nature Communications

Article Title: Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

doi: 10.1038/s41467-022-32311-2

Figure Lengend Snippet: a MES28 GSCs were subjected to RNA immunoprecipitation using an anti-DDX3 antibody or IgG (negative control). Immunoprecipitates were analysed by RT-qPCR with specific primers for the circEZH2 IRES sequence ( P = 3.35e−05), GAPDH (negative control, P = 0.0636) and BCL2 (positive control, P = 7.09e−04). b DDX3 and circEZH2 RNA levels in MES28 and GSC23 cells transfected with DDX3 siRNAs. MES28, si-DDX3-1, P = 8.66e−10, si-DDX3-2, P = 9.31e−08; GSC23, si-DDX3-1, P = 5.77e−05, si-DDX3-2, P = 4.06e−05. c Protein levels of DDX3 and EZH2-92aa in MES28 and GSC23 cells transfected with DDX3 siRNAs. d Expression of DDX3 in GBM datasets from TCGA (GBM and LGG dataset, plotted by GEPIA2, [ http://gepia.cancer-pku.cn/ ]) and CGGA (dataset mRNAseq_325, [ http://www.cgga.org.cn/index.jsp ]). Data are presented as boxes containing the median (centre line), the first and third quartiles (box limits). The whiskers indicate the maxima and minima. One-way ANOVA test. For TCGA dataset, * P < 0.01. For CGGA dataset, P = 0.00078. e Protein levels of DDX3 and EZH2-92aa in NHAs and several patient-derived GSC lines. The data in ( a ), ( b ), ( c ) and ( e ) are pooled from three independent experiments. The data are presented as the mean ± SD. Unpaired two-tailed Student’s t test was used to determine the significance of differences between the indicated groups. * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source data file.

Article Snippet: A rabbit polyclonal antibody specific for EZH2-92aa (1:500) was produced by GenScript Biotech (Jiangsu, China).

Techniques: RNA Immunoprecipitation, Negative Control, Quantitative RT-PCR, Sequencing, Positive Control, Transfection, Expressing, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

doi: 10.1038/s41467-022-32311-2

Figure Lengend Snippet: a Left, representative IF image of MES28 GSCs transfected with EZH2-92aa-3×flag and stained with an anti-flag antibody. VA, vector alone. Scale bar, 5 μm. Right, immunoblot of EZH2-92aa-3×Flag or endogenous EZH2-92aa in the indicated cellular fraction of MES28 GSCs transfected with EZH2-92aa-3×Flag or GSC23 GSCs using an anti-Flag antibody or the custom anti-EZH2-92aa antibody. W, whole cell lysate; N, nuclear fraction; C, cytoplasmic fraction. b Left, mRNA levels of MICA/MICB in circEZH2 stable KD MES28 GSCs. Right, immunoblot showing the levels of MICA/MICB in circEZH2 stable KD MES28 GSCs. Scramble vs sh-circEZH2, circEZH2 P = 0.0023, MICA P = 0.0001, MICB P = 0.0046. c , d Luciferase activity driven by MICA/MICB in MES28 GSCs transfected with increasing doses of the EZH2-92aa OV plasmid. MICA, 0 vs 2 P = 0.0078, 0 vs 5 P = 0.0002, 0 vs 10 P = 0.0002; MICB, 0 vs 2 P = 0.0042, 0 vs 5 P = 8.14e−06, 0 vs 10 P = 5.25e−06. e , f Luciferase activity of the MICA/MICB promoter fragments fused to a luciferase reporter gene. MICA, vector 1# vs EZH2-92aa 1# P = 1.49e−04, vector 1# vs EZH2-92aa 2# P = 6.92e−05, vector 1# vs EZH2-92aa 3# P = 2.11e−04; MICB, vector 1# vs EZH2-92aa 1# P = 0.0014, vector 1# vs EZH2-92aa 2# P = 0.0012, vector 1# vs EZH2-92aa 3# P = 0.0003, vector 1# vs EZH2-92aa 4# P = 0.0006. g , h ChIP-qPCR analysis of the binding site of EZH2-92aa-3×Flag in the MICA/MICB promoters in MES28 GSCs transfected with EZH2-92aa-3×Flag. IgG vs Flag, MICA P = 0.0077, MICB P = 0.0161. i , j EMSA was performed using the nuclear extract of MES28 GSCs transfected with EZH2-92aa-3×Flag and 6 specific biotin-labelled MICA/MICB probes. Independent experiments were performed three times with similar results. k , l EMSA was performed using the nuclear extract of MES28 GSCs transfected with EZH2-92aa-3×Flag, biotin-labelled MICA/MICB probes, a 200-fold excess of unlabelled MICA/MICB probes (200× WT competitor), biotin-labelled mutated MICA/MICB probes (200× Mut competitor) and an anti-flag antibody. Independent experiments were performed three times with similar results. m , n Mutants of the EZH2-92aa binding site in the MICA/MICB promoter were constructed. Luciferase activity of the WT or mutated MICA/MICB constructs with EZH2-92aa-3×Flag OV. Ctrl vs 92aa, MICA WT, P = 5.52e−05; MICB WT, P = 1.40e−05. The data are pooled from three independent experiments. The data are presented as the mean ± SD. Unpaired two-tailed Student’s t test was used to determine the significance of differences between the indicated groups where applicable. ns, nonsignificant, * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source data file.

Article Snippet: A rabbit polyclonal antibody specific for EZH2-92aa (1:500) was produced by GenScript Biotech (Jiangsu, China).

Techniques: Transfection, Staining, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, ChIP-qPCR, Binding Assay, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

doi: 10.1038/s41467-022-32311-2

Figure Lengend Snippet: a Relative RNA levels of ULBPs (ULBP1-6) in circEZH2 stable KD MES28 GSCs. Scramble vs sh-circEZH2, circEZH2 P = 1.68e−04, ULBP1 P = 2.49e−04, ULBP4 P = 8.36e−04, ULBP6 P = 6.42e−05. b Relative RNA and protein levels of circEZH2/EZH2-92aa and EZH2 were measured in MES28 and GSC23 GSCs transfected with circEZH2-siRNAs or control scrambled siRNAs. H3K27me3 protein levels were also determined. MES28, scramble vs si-circEZH2-1, P = 1.87e−08, si-circEZH2-2, P = 9.90e−09; GSC23, scramble vs si-circEZH2-1, P = 6.00e−05, si-circEZH2-2, P = 3.25e−05. c Left, half-life of EZH2 in circEZH2-siRNA-transfected MES28 GSCs. Right, quantitative analysis of the immunoblotting data by greyscale analysis at the indicated time points. CHX = cycloheximide. Scramble vs sh-circEZH2, 3 h P = 0.0025, 6 h P = 0.0015, 9 h P = 0.0069. d Protein level of EZH2 in circEZH2-siRNA-transfected MES28 and GSC23 GSCs after MG132 treatment (20 μM) for 6 h. e 293T cells were transfected with EZH2-92aa-3xflag, and total cell lysates were subjected to immunoprecipitation using anti-Flag and anti-FBXW7 antibodies, followed by immunoblotting with an anti-Flag or anti-FBXW7 antibody. f EZH2-92aa-3xFlag and FBXW7-6xHis were transfected into 293T cells as indicated in combination with Ub-HA. After treatment with MG132 (20 μM) for 6 h, total cell lysates were subjected to immunoprecipitation using an anti-EZH2 antibody, followed by immunoblotting with an anti-HA antibody. g MES28 and GSC23 circEZH2 stable KD or control cells were analysed for H3K27me3 levels in the ULBP1 promoter using a ChIP assay. Relative fold changes compared with IgG are shown. Scramble anti-H3K27me3 vs sh-circEZH2 anti-H3K27me3, MES28 P = 3.07e−04, GSC23 1.15e−04. h Relative RNA levels of ULBP1 in MES28 and GSC23 GSCs transfected with scrambled shRNA control, sh-circEZH2 or sh-circEZH2 together with the EZH2 OV plasmid. Scramble vs sh-circEZH2, MES28 P = 3.25e−05, GSC23 P = 0.0067; sh-circEZH2 vs sh-circEZH2+OV-EZH2, MES29 P = 2.65e−04, GSC23 P = 0.0157. i Cytotoxicity of NK cells after coculture with MES28 GSCs with stable circEZH2 KD, stable circEZH2 KD plus NKG2D block (10 μg/ml). Sh-circEZH2 vs sh-circEZH2 plus block, NK-92MI, 1:2 P = 0.0481, 1:5 P = 0.0147, 1:10 P = 0.0092; NK #1, 1:2 P = 0.0238, 1:5 P = 0.0155, 1:10 P = 0.0097; NK #2, 1:2 P = 0.0231, 1:5 P = 0.0086, 1:10 P = 0.0087. j Left panel, images showing remaining live MES28 GSCs with indicated modifications after coculture with NK cells using calcein AM staining. NKG2D block (10 μg/ml) was added in the indicated groups. Scale bar, 50 μm. Right panel, quantification of the remaining live GSCs. Sh-circEZH2 vs sh-circEZH2 plus block, NK-92MI, P = 0.0051; NK #1, P = 0.0081; NK #2, P = 0.0059. The data are presented as the mean ± SD from three independent experiments. Unpaired two-tailed Student’s t test was used to determine the significance of differences between the indicated groups where applicable. ns, nonsignificant, * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source data file.

Article Snippet: A rabbit polyclonal antibody specific for EZH2-92aa (1:500) was produced by GenScript Biotech (Jiangsu, China).

Techniques: Transfection, Control, Western Blot, Immunoprecipitation, shRNA, Plasmid Preparation, Blocking Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

doi: 10.1038/s41467-022-32311-2

Figure Lengend Snippet: The diagram illustrates the mechanism underlying EZH2-92aa-mediated immune escape of GSCs from NK cells. EZH-92aa, encoded by circEZH2 in GSCs, can not only directly bind to the promoters of MICA/B and represses their transcription, but also serves as a decoy of E3-ligase FBXW7 to protect EZH2 from degradation and indirectly impedes the expression of ULBPs. The downregulation of NKG2DLs (MICA/B and ULBPs) caused by EZH2-92aa is responsible for the immune evasion of GSCs from NK cytotoxicity.

Article Snippet: A rabbit polyclonal antibody specific for EZH2-92aa (1:500) was produced by GenScript Biotech (Jiangsu, China).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: EZH2 and HDAC1/2 expression in PTCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Representative immunohistochemical features of HDAC1 (left), HDAC2 (middle), and EZH2 (right) in PTCL-NOS, ALCL, NK/T and AITL. All images were captured at ×200 and ×400 magnifications. HDAC, histone deacetylase; EZH2, enhancer of zeste homolog 2; PTCL-NOS, peripheral T cell lymphoma not otherwise specified; ALCL, anaplastic large cell lymphoma; NK/T, natural killer/T-cell; AITL, angioimmunoblastic T-cell lymphoma.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Immunohistochemical staining, Histone Deacetylase Assay

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between EZH2 and HDAC1/2 in four subtypes of PTCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques:

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between EZH2/HDAC1/2 expression and the clinicopathological characteristics in PTCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between the EZH2/HDAC1/2 expression and the clinicopathological characteristics in PTCL-NOS.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between the EZH2/HDAC1/2 expression and the clinicopathological characteristics in NK/TCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: OS rates based upon the expression levels of EZH2 and HDAC1/2 in PTCL. (A) Significant trend towards poorer OS rates for patients with high EZH2 expression (P=0.012). (C) High expression of HDAC2 was correlated with a poorer OS rate compared with low expression of HDAC2 (P<0.001), which was not observed in the HDAC1 group [(B); P=0.353]. OS, overall survival; HDAC, histone deacetylase; EZH2, enhancer of zeste homolog 2.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing, Histone Deacetylase Assay

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: OS based upon the levels of EZH2 and HDAC1/2 in PTCL-NOS. (A) Significant trend towards poorer OS rates for patients with high EZH2 (P=0.002). (C) High expression of HDAC2 was correlated with a poorer OS rate, compared with low expression of HDAC2 (P<0.001), which was not observed in the HDAC1 group [(B); P=0.339]. OS, overall survival; HDAC, histone deacetylase; EZH2, enhancer of zeste homolog 2; PTCL-NOS, peripheral T cell lymphoma not otherwise specified.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing, Histone Deacetylase Assay