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. 2015 Sep 25:6:8494.
doi: 10.1038/ncomms9494.

EZH2-mediated loss of miR-622 determines CXCR4 activation in hepatocellular carcinoma

Haiou Liu  1 , Yidong Liu  2 , Weisi Liu  2 , Weijuan Zhang  3 , Jiejie Xu  2
Affiliations

EZH2-mediated loss of miR-622 determines CXCR4 activation in hepatocellular carcinoma

Haiou Liu et al. Nat Commun. .

Erratum in

Abstract

The CXC chemokine receptor 4 (CXCR4) exerts a variety of functions at different steps of hepatocellular carcinoma (HCC) progression. The molecular mechanisms and therapeutic value of CXCR4 in the development of HCC remain undefined. Here we show that aberrant CXCR4 overexpression is associated with poor prognosis and aggressive characteristics of HCC. Suppression of CXCR4 activity via CXCR4 knockdown, AMD3100 or neutralizing antibody administration inhibits hepatoma cell tumorigenesis in vitro and in vivo. CXCR4 overexpression displays the opposite effects. Using Mir library screening we identify miR-622 as a regulator of CXCR4. Further studies show that miR-622 directly target the 3' untranslated region of CXCR4 and is transcriptionally repressed by EZH2-induced H3K27 trimethylation and promoter methylation. EZH2/miR-622 promotes tumorigenesis through CXCR4. EZH2-mediated loss of miR-622 is found to correlate with CXCR4 overexpression and unfavourable prognosis in HCC patients. This study establishes EZH2/miR-622/CXCR4 as a potential adverse prognostic factor and therapeutic target for HCC patients.

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Figures

Figure 1
Figure 1. Upregulation of CXCR4 in HCC correlated with poor patient survival.
(a) Representative immunohistochemistry (IHC) staining with CXCR4 (scale bar, 50 μm). (b) Scatter plots for corresponding evaluated IHC score in tumour and peritumour HCC specimens (n=127). The horizontal lines in the plots represent the median and the interquartile range. The P values were calculated using Student's t-test. (c) Western blot analysis of CXCR4, GAPDH in cytoplasmic extracts (Cyto) and CXCR4, lamin B1 in nuclear extracts (NE) in HCC specimens. Data are representative immunoblots of three independent assays. (d) Representative IHC staining with CXCR4 from TNM stage I to III of HCC specimens (scale bar, 50 μm)(left panel). Scatter plots for corresponding evaluated IHC score from TNM stage I to III of HCC specimens (right panel). Error bars represent mean±s.d. IHC scores were compared by one-way analysis of variance and Student's t-test, *P<0.05. (e,f) Kaplan–Meier plots indicate the overall survival (e) and RFS (f) for HCC patients categorized by CXCR4 expression (n=63 for high-CXCR4 group versus n=64 for low-CXCR4 group), P value is determined by log-rank test.
Figure 2
Figure 2. CXCR4 promotes hepatoma cell proliferation and survival in vitro.
(a) Western blot analysis of CXCR4 and GAPDH for Huh7, PLC/PRF/5, Hep3B, HepG2, SNU448 and SK-Hep1 cells (upper panel), for Huh7 cells stably transfected with empty vector or CXCR4 (lower left panel) and for SK-Hep1 cells stably transfected with nonspecific shRNA (shCon) or CXCR4-specific shRNA (shCXCR4) (lower right panel). Data are representative immunoblots of three independent assays. (b) Cell proliferation analysis for Huh7 cells without or with stably CXCR4 overexpression, SK-Hep1 cells without or with stably CXCR4 knockdown, SK-Hep1 cells without or with AMD3100 (100 nM) or CXCR4-neutralization antibody (100 μg ml−1) treatment (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (c) Representative micrographs and quantification of soft agar colonies for above-mentioned hepatoma cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (d) Representative micrographs and quantification of the above-mentioned hepatoma cells in the Transwell migration assay (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (e) Representative dot plots of flow cytometric analysis of above-mentioned hepatoma cells cultured in serum-deprivation medium for 48 h. Cells were subjected to annexin-V and Propidium lodide (PI) staining, and the percentage of cells displaying annexin-V single positive and annexin-V, PI double positive are denoted (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d.
Figure 3
Figure 3. CXCR4 promotes hepatoma cells growth in vivo.
(a) Tumour growth curve and quantification of weight of the tumours that developed in nude mice injected subcutaneously with Huh7-control or Huh7-CXCR4 cells (n=10). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (b) Tumour growth curve and quantification of weight of the tumours that developed in nude mice injected subcutaneously with SK-Hep1-shCon or SK-Hep1-shCXCR4 cells (n=10). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (c) Tumour growth curve of mice bearing SK-Hep1 xenografts following intratumoural administration of PBS or 10 mg kg−1 AMD3100 (n=10). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (left panel). Kaplan–Meier survival analysis was performed for above-mentioned xenograft mice (n=10), P value is determined by log-rank test (right panel). (d) Tumour growth curve analysis of mice bearing SK-Hep1 xenografts following administration of 1 mg IgG or anti-CXCR4 neutralization antibody (n=10). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (left panel). Kaplan–Meier survival analysis was performed for above-mentioned xenograft mice (n=10), P value is determined by log-rank test (right panel). (e) Representative IHC staining of Ki67 for SK-Hep1 xenografts with indicated administrations (scale bar, 50 μm; upper panel). Quantification of the percentage of Ki67-positive cells (n=10, lower panel). Student's t-test, *P<0.05. Error bars in panels are defined as s.d.
Figure 4
Figure 4. CXCR4 is a direct miR-622 target.
(a) Schema of the candidate miRNAs by different prediction algorithms. Each labelled circle represents one prediction algorithm with the number of its predicted miRNAs, and the number listed in overlapping of circles is simultaneously predicted by different algorithms. (b) Schematic model for miRNA screening to target CXCR4. (c) Heatmap obtained from RT–PCR of HCC and corresponding peritumour specimens. Each column represents the average of three biological replicates. The relative high expression is indicated in red, whereas the relative low expression is in green. (d) Western blot analysis of CXCR4 and GAPDH for SK-Hep1 cells transiently transfected with miR-nc, miR-302c, miR-139-5p, miR-9, miR-206 and miR-622 mimic (left panel) and for Huh7 cells transiently transfected with anti-miR-nc, anti-miR-302c, anti-miR-139-5p, anti-miR-9, anti-miR-206 and anti-miR-622 (right panel). Data are representative immunoblots of three independent assays. (e) Sequences of miR-622 and the potential miR-622-binding sites at the 3′-UTR of CXCR4. Also shown are nucleotides mutated in CXCR4-3′-UTR mutant. Seed sequences are marked. (f) Luciferase activity assay for pGL3-CXCR4 3′-UTR (wt) or pGL3-CXCR4 3′-UTR (mut) relative to Renilla luciferase activity for SK-Hep1 and SNU448 cells transiently transfected with miR-nc or miR-622-mimc (n=3, upper panel). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. Western blot analysis of CXCR4 and GAPDH for SK-Hep1 and SNU448 cells transiently transfected with miR-nc and miR-622-mimc (lower panel). Data are representative immunoblots of three independent assays. (g) Luciferase activity assay for pGL3-CXCR4 3′-UTR (wt) or pGL3-CXCR4 3′-UTR (mut) relative to Renilla luciferase activity for PLC/PRF/5 and Huh7 cells transiently transfected with anti-miR-nc or anti-miR-622 (n=3, upper panel). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. Western blot analysis of CXCR4 and GAPDH for PLC/PRF/5 and Huh7 cells transiently transfected with anti-miR-nc or anti-miR-622 (lower panel). Data are representative immunoblots of three independent assays.
Figure 5
Figure 5. CXCR4 mediate the effects of miR-622 on hepatoma cell growth and migration.
Hepatoma cells were transfected with anti-miR-622 or miR-622 before transfection with either CXCR4-siRNA or CXCR4. (a) Immunoblot analysis showing the inhibition of anti-miR-622-induced upregulation of CXCR4 by CXCR4-siRNA in Huh7 and PLC/PRF/5 cells, and showing the rescue of miR-622-induced downregulation of CXCR4 by CXCR4 in SK-Hep1 and SNU448 cells. Data are representative immunoblots of three independent assays. (b) Growth assay showing that CXCR4 inhibition partially reverse the proliferative effects of anti-miR-622 promotion in Huh7 and PLC/PRF/5 cells, and CXCR4 overexpression partially rescues the proliferative effects of miR-622 inhibition in SK-Hep1 and SNU448 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (c) Transwell migration assay showing that CXCR4 inhibition partially reverse the migration effects of anti-miR-622 promotion in Huh7 and PLC/PRF/5 cells, and CXCR4 overexpression partially rescues the proliferative effects of miR-622 inhibition in SK-Hep1 and SNU448 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d.
Figure 6
Figure 6. DNA methylation status of the miR-622 promoter.
(a) Scheme depicting the genomic localization of miR-622. TSS is indicated with an arrow. Transcription start site (TSS) locations were predicted using miRStart (http://mirstart.mbc.nctu.edu.tw/). The regions analysed by BSP are indicated. (b) The sequence level detail of the miR-622 promoter regions (−3,616 to −3,403, 213 bp); the CpG dinucleotides within this region are numbered as 1–14. (c) The methylation status of the miR-622 promoter in the normal liver tissue (normal) and SK-Hep1, SNU448, HepG2, Hep3b, PLC/PRF/5 and Huh7 cell lines. The open and filled circles indicate the unmethylated and methylated CpGs, respectively. (d) Quantitative PCR (qPCR) analysis of miR-622 expression in indicated cells treated with 5 μmol 5-aza-dC for 72 h (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (e) The methylation status of the miR-622 promoter in 13 pairs of HCC and peritumour tissues. (f) qPCR analysis of miR-622 expression in 13 pairs of HCC and peritumour tissues (left) and in HCC tissues with or without methylation in miR-622 promoter (right; n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d.
Figure 7
Figure 7. Expression of miR-622 is modulated with EZH2 in HCC cells.
(a) Western blot analysis of EZH2, CXCR4, H3K27me3, H3K9me3, Histone 3 and GAPDH (left panel), quantitative PCR (qPCR) analysis of miR-622 (right panel) for Huh7 cells stably transfected with empty vector or EZH2 (n=3). Data are representative immunoblots of three independent assays. Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (b) CHIP–qPCR analysis of EZH2, H3K27me3 and H3K9me3 enrichment at upstream of miR-622 for above-mentioned Huh7 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (c) Western blot analysis of EZH2, CXCR4, H3K27me3, H3K9me3, Histone 3 and GAPDH (left panel), qPCR analysis of miR-622 (right panel) for SK-Hep1 cells stably transfected with nonspecific shRNA (shCon) or EZH2-specific shRNA (shEZH2; n=3). Data are representative immunoblots of three independent assays. Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (d) CHIP–qPCR analysis of EZH2, H3K27me3 and H3K9me3 enrichment at upstream of miR-622 for above-mentioned SK-Hep1 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (e) Western blot analysis of EZH2, CXCR4, H3K27me3, H3K9me3, Histone 3 and GAPDH (left panel), qPCR analysis of miR-622 (right panel) for SK-Hep1 cells without or with DZNep (10 μM) treatment for 72 h (n=3). Data are representative immunoblots of three independent assays. Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (f) CHIP–qPCR analysis of EZH2, H3K27me3 and H3K9me3 enrichment at upstream of miR-622 for SK-Hep1 cells without or with DZNep treatment (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d.
Figure 8
Figure 8. EZH2 regulates CXCR4 by controlling miR-622 expression.
(a) Quantitative PCR analysis of miR-622 for Huh7 cells cotransfected with or without miR-622 and CXCR4, and SK-Hep1 cells cotransfected with or without EZH2-shRNA and anti-miR-622 inhibitor (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (b) Transwell migration assay showing that miR-622 partially reverse the migration effect of EZH2 promotion in Huh7 cells, and anti-miR-622 inhibitor partially rescues the migration effect of EZH2-shRNA inhibition in SK-Hep1 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (c) Growth assay showing that miR-622 partially reverse the proliferative effects of EZH2 promotion in Huh7 cells, and anti-miR-622 inhibitor partially rescues the proliferative effects of EZH2-shRNA inhibition in SK-Hep1 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (d) Western blot analysis of CXCR4, EZH2 and GAPDH in SK-Hep1 cells stably transfected with EZH2-shRNA cotransfected with or without CXCR4. Data are representative immunoblots of three independent assays. (e) Transwell migration assay showing that CXCR4 overexpression partially rescues the proliferative effects of EZH2-shRNA inhibition in SK-Hep1 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d. (f) Growth assay showing that CXCR4 partially rescues the proliferative effects of EZH2-shRNA inhibition in SK-Hep1 cells (n=3). Student's t-test, *P<0.05. Error bars in panels are defined as s.d.
Figure 9
Figure 9. EZH2/miR-622/CXCR4 pathway correlates with poor prognosis in HCC patients.
(a) Representative immunohistochemistry (IHC) staining with CXCR4, EZH2 and miRNA in situ hybridization (ISH) staining with miR-622 (scale bar, 50 μm). (b) Scatter plots showing the negative correlation between CXCR4 IHC score and miR-622 ISH score, and positive correlation between CXCR4 and EZH2 IHC score in HCC. Pearson's coefficient tests were performed to assess statistical significance. (c) Kaplan–Meier overall survival analysis curve (upper panel) or RFS analysis curve (lower panel) is shown for high- or low-risk survival group in 127 HCC patients. High CXCR4 expression and simultaneously low miR-622 level are significantly associated with both poorest overall survival and RFS. P value is determined by log-rank test. (d) Proposed model for CXCR4 upregulation in hepatoma cells. EZH2 are linked to CXCR4 activation via miR-622 regulation.
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