Zinc fingers and homeoboxes 2 inhibits hepatocellular carcinoma cell proliferation and represses expression of Cyclins A and E

Abstract

Background & aims: Zinc-fingers and homeoboxes 2 (ZHX2) represses transcription of several genes associated with liver cancer. However, little is known about the role of ZHX2 in the development of hepatocellular carcinoma (HCC). We investigated the mechanisms by which ZHX2 might affect proliferation of HCC cells.

Methods: We overexpressed and knocked down ZHX2 in HCC cells and analyzed the effects on proliferation, colony formation, and the cell cycle. We also analyzed the effects of ZHX2 overexpression in growth of HepG2.2.15 tumor xenografts in nude mice. Chromatin immunoprecipitation and luciferase reporter assays were used to measure binding of ZHX2 target promoters. Levels of ZHX2 in HCC samples were evaluated by immunohistochemistry.

Results: ZHX2 overexpression significantly reduced proliferation of HCC cells and growth of tumor xenografts in mice; it led to G1 arrest and reduced levels of Cyclins A and E in HCC cell lines. ZHX2 bound to promoter regions of CCNA2 (which encodes Cyclin A) and CCNE1 (which encodes Cyclin E) and inhibited their transcription. Knockdown of Cyclin A or Cyclin E reduced the increased proliferation mediated by ZHX2 knockdown. Nuclear localization of ZHX2 was required for it to inhibit proliferation of HCC cells in culture and in mice. Nuclear localization of ZHX2 was reduced in human HCC samples, even in small tumors (diameter, <5 cm), compared with adjacent nontumor tissues. Moreover, reduced nuclear levels of ZHX2 correlated with reduced survival times of patients, high levels of tumor microvascularization, and hepatocyte proliferation.

Conclusions: ZHX2 inhibits HCC cell proliferation by preventing expression of Cyclins A and E, and reduces growth of xenograft tumors in mice. Loss of nuclear ZHX2 might be an early step in the development of HCC.

Figure 1. Elevated ZHX2 levels inhibit growth of HCC both in vitro (A,B) and in vivo (C,D,E,F)

(A) Proliferation of HepG2.2.15 and HepG2 cells transfected with pcZHX2 or control pcDNA3.0 and SMMC7721 and QS7701 cells transfected with shRNAs pS2360 or pS1674. Cells were assayed over a 4-day period and data shown is mean±SD from four experiments. ZHX2 and β-actin levels in cell lines were determined by western blot. (B) Colony formation HCC cell lines transfected with pcZHX2 or ZHX2 shRNAs as described in 1A. Plates are shown on the left; the statistical results are shown in the right (Mean±SD from three experiments) * * *, p<0.001. (C) Real-time RT-PCR analysis of ZHX2 mRNA in pcZHX2-injected tumors and control pcDNA3.0-injected tumors. (Mean ±SD, n=6). * *, p<0.01. (D) Xenograft tumor growth determined over a 20 day period. (Mean±SD, n=6); *, p<0.05, * * *, p<0.001. (E) Weight of pcZHX2- and pcDNA3.0-injected tumors at time of sacrifice. (Mean±SD, n=6); *, p<0.05. Images of tumors from each group is shown on top. (F) Immunohistochemical staining of Ki-67 of pcDNA3.0-and pcZHX2-injected tumors; statistical results were shown in the bottom. (Mean±SD, n=6); * * *, p<0.001.

Figure 1. Elevated ZHX2 levels inhibit growth of HCC both in vitro (A,B) and in vivo (C,D,E,F)

(A) Proliferation of HepG2.2.15 and HepG2 cells transfected with pcZHX2 or control pcDNA3.0 and SMMC7721 and QS7701 cells transfected with shRNAs pS2360 or pS1674. Cells were assayed over a 4-day period and data shown is mean±SD from four experiments. ZHX2 and β-actin levels in cell lines were determined by western blot. (B) Colony formation HCC cell lines transfected with pcZHX2 or ZHX2 shRNAs as described in 1A. Plates are shown on the left; the statistical results are shown in the right (Mean±SD from three experiments) * * *, p<0.001. (C) Real-time RT-PCR analysis of ZHX2 mRNA in pcZHX2-injected tumors and control pcDNA3.0-injected tumors. (Mean ±SD, n=6). * *, p<0.01. (D) Xenograft tumor growth determined over a 20 day period. (Mean±SD, n=6); *, p<0.05, * * *, p<0.001. (E) Weight of pcZHX2- and pcDNA3.0-injected tumors at time of sacrifice. (Mean±SD, n=6); *, p<0.05. Images of tumors from each group is shown on top. (F) Immunohistochemical staining of Ki-67 of pcDNA3.0-and pcZHX2-injected tumors; statistical results were shown in the bottom. (Mean±SD, n=6); * * *, p<0.001.

Figure 1. Elevated ZHX2 levels inhibit growth of HCC both in vitro (A,B) and in vivo (C,D,E,F)

(A) Proliferation of HepG2.2.15 and HepG2 cells transfected with pcZHX2 or control pcDNA3.0 and SMMC7721 and QS7701 cells transfected with shRNAs pS2360 or pS1674. Cells were assayed over a 4-day period and data shown is mean±SD from four experiments. ZHX2 and β-actin levels in cell lines were determined by western blot. (B) Colony formation HCC cell lines transfected with pcZHX2 or ZHX2 shRNAs as described in 1A. Plates are shown on the left; the statistical results are shown in the right (Mean±SD from three experiments) * * *, p<0.001. (C) Real-time RT-PCR analysis of ZHX2 mRNA in pcZHX2-injected tumors and control pcDNA3.0-injected tumors. (Mean ±SD, n=6). * *, p<0.01. (D) Xenograft tumor growth determined over a 20 day period. (Mean±SD, n=6); *, p<0.05, * * *, p<0.001. (E) Weight of pcZHX2- and pcDNA3.0-injected tumors at time of sacrifice. (Mean±SD, n=6); *, p<0.05. Images of tumors from each group is shown on top. (F) Immunohistochemical staining of Ki-67 of pcDNA3.0-and pcZHX2-injected tumors; statistical results were shown in the bottom. (Mean±SD, n=6); * * *, p<0.001.

Figure 1. Elevated ZHX2 levels inhibit growth of HCC both in vitro (A,B) and in vivo (C,D,E,F)

(A) Proliferation of HepG2.2.15 and HepG2 cells transfected with pcZHX2 or control pcDNA3.0 and SMMC7721 and QS7701 cells transfected with shRNAs pS2360 or pS1674. Cells were assayed over a 4-day period and data shown is mean±SD from four experiments. ZHX2 and β-actin levels in cell lines were determined by western blot. (B) Colony formation HCC cell lines transfected with pcZHX2 or ZHX2 shRNAs as described in 1A. Plates are shown on the left; the statistical results are shown in the right (Mean±SD from three experiments) * * *, p<0.001. (C) Real-time RT-PCR analysis of ZHX2 mRNA in pcZHX2-injected tumors and control pcDNA3.0-injected tumors. (Mean ±SD, n=6). * *, p<0.01. (D) Xenograft tumor growth determined over a 20 day period. (Mean±SD, n=6); *, p<0.05, * * *, p<0.001. (E) Weight of pcZHX2- and pcDNA3.0-injected tumors at time of sacrifice. (Mean±SD, n=6); *, p<0.05. Images of tumors from each group is shown on top. (F) Immunohistochemical staining of Ki-67 of pcDNA3.0-and pcZHX2-injected tumors; statistical results were shown in the bottom. (Mean±SD, n=6); * * *, p<0.001.

Figure 1. Elevated ZHX2 levels inhibit growth of HCC both in vitro (A,B) and in vivo (C,D,E,F)

(A) Proliferation of HepG2.2.15 and HepG2 cells transfected with pcZHX2 or control pcDNA3.0 and SMMC7721 and QS7701 cells transfected with shRNAs pS2360 or pS1674. Cells were assayed over a 4-day period and data shown is mean±SD from four experiments. ZHX2 and β-actin levels in cell lines were determined by western blot. (B) Colony formation HCC cell lines transfected with pcZHX2 or ZHX2 shRNAs as described in 1A. Plates are shown on the left; the statistical results are shown in the right (Mean±SD from three experiments) * * *, p<0.001. (C) Real-time RT-PCR analysis of ZHX2 mRNA in pcZHX2-injected tumors and control pcDNA3.0-injected tumors. (Mean ±SD, n=6). * *, p<0.01. (D) Xenograft tumor growth determined over a 20 day period. (Mean±SD, n=6); *, p<0.05, * * *, p<0.001. (E) Weight of pcZHX2- and pcDNA3.0-injected tumors at time of sacrifice. (Mean±SD, n=6); *, p<0.05. Images of tumors from each group is shown on top. (F) Immunohistochemical staining of Ki-67 of pcDNA3.0-and pcZHX2-injected tumors; statistical results were shown in the bottom. (Mean±SD, n=6); * * *, p<0.001.

Figure 1. Elevated ZHX2 levels inhibit growth of HCC both in vitro (A,B) and in vivo (C,D,E,F)

(A) Proliferation of HepG2.2.15 and HepG2 cells transfected with pcZHX2 or control pcDNA3.0 and SMMC7721 and QS7701 cells transfected with shRNAs pS2360 or pS1674. Cells were assayed over a 4-day period and data shown is mean±SD from four experiments. ZHX2 and β-actin levels in cell lines were determined by western blot. (B) Colony formation HCC cell lines transfected with pcZHX2 or ZHX2 shRNAs as described in 1A. Plates are shown on the left; the statistical results are shown in the right (Mean±SD from three experiments) * * *, p<0.001. (C) Real-time RT-PCR analysis of ZHX2 mRNA in pcZHX2-injected tumors and control pcDNA3.0-injected tumors. (Mean ±SD, n=6). * *, p<0.01. (D) Xenograft tumor growth determined over a 20 day period. (Mean±SD, n=6); *, p<0.05, * * *, p<0.001. (E) Weight of pcZHX2- and pcDNA3.0-injected tumors at time of sacrifice. (Mean±SD, n=6); *, p<0.05. Images of tumors from each group is shown on top. (F) Immunohistochemical staining of Ki-67 of pcDNA3.0-and pcZHX2-injected tumors; statistical results were shown in the bottom. (Mean±SD, n=6); * * *, p<0.001.

Figure 2. ZHX2 induces G1 arrest and represses Cyclin A and Cyclin E expression

(A) HepG2 cells transfected with pcZHX2 or pcDNA3.0 or (B) SMMC7721 cells transfected with scrambled control vector or pS1764 or pS2360 shRNA vectors were analyzed by PI staining and flow cytometry. A representative plot from one experiment and Mean±SD from three experiments are shown. (C) Western blot to monitor levels of ZHX2, Cyclin A, Cyclin D1, Cyclin E, p21, p27 and β-actin in transfected HCC cell lines described in (A) and (B). The experiments were repeated for four times and one representative result is shown.

Figure 2. ZHX2 induces G1 arrest and represses Cyclin A and Cyclin E expression

(A) HepG2 cells transfected with pcZHX2 or pcDNA3.0 or (B) SMMC7721 cells transfected with scrambled control vector or pS1764 or pS2360 shRNA vectors were analyzed by PI staining and flow cytometry. A representative plot from one experiment and Mean±SD from three experiments are shown. (C) Western blot to monitor levels of ZHX2, Cyclin A, Cyclin D1, Cyclin E, p21, p27 and β-actin in transfected HCC cell lines described in (A) and (B). The experiments were repeated for four times and one representative result is shown.

Figure 2. ZHX2 induces G1 arrest and represses Cyclin A and Cyclin E expression

(A) HepG2 cells transfected with pcZHX2 or pcDNA3.0 or (B) SMMC7721 cells transfected with scrambled control vector or pS1764 or pS2360 shRNA vectors were analyzed by PI staining and flow cytometry. A representative plot from one experiment and Mean±SD from three experiments are shown. (C) Western blot to monitor levels of ZHX2, Cyclin A, Cyclin D1, Cyclin E, p21, p27 and β-actin in transfected HCC cell lines described in (A) and (B). The experiments were repeated for four times and one representative result is shown.

Figure 3. ZHX2 binds to and represses activity of the Cyclin A and Cyclin E promoters

(A) Real-time RT-PCR analysis of ZHX2, Cyclin A and Cyclin E mRNA levels in HepG2 cells transfected with pcZHX2 or pcDNA3.0 or SMMC7721 cells transfected with shRNAs. Data (mean±SD) of three experiments are shown. *, p<0.05, * *, p<0.01, * * *, p<0.001. (B) Inhibition of the pGL3-Ep and pGL3-Ap luciferase reporter genes by ZHX2 in HepG2 cells. Data (mean±SEM) of four experiments are shown. * *, p<0.01. (C) Diagram of the Cyclin E and Cyclin A promoters, extending to -402 and -505, respectively, including the NF-Y site in the cyclin A promoter and location of the primers (solid or dotted lines under promoters) used for ChIP analysis. (D) ChIP analysis of DNA from HepG2 cells transfected with ZHX2-HA. PCR amplification of HA-immunoprecipitated DNA using the primers shown in (C) shows ZHX2 binding to Cyclin A and Cyclin E promoters but not Cyclin D1. One of three independent experiments is shown. (E) Colony formation of QSG7701 and SMMC7721 cells transfected with shRNAs against ZHX2 (pS1674 or pS2360) and siRNAs agaist Cyclin A (sicycA) or Cyclin E (sicycE). One representative plate of each group is shown on the left; results from three independent experiments are shown in the right. (mean±SD) * * *, p<0.001. (F) Proliferation of QSG7701 and SMMC7721 cells that were co-transfected with ZHX2 shRNAs along with siRNAs for Cyclin A, Cyclin E or Cyclin D1. Cells were measured using CCK-8 48h after cotransfection. Mean±SD of three experiments is shown. ; * * *, p<0.001.

Figure 3. ZHX2 binds to and represses activity of the Cyclin A and Cyclin E promoters

(A) Real-time RT-PCR analysis of ZHX2, Cyclin A and Cyclin E mRNA levels in HepG2 cells transfected with pcZHX2 or pcDNA3.0 or SMMC7721 cells transfected with shRNAs. Data (mean±SD) of three experiments are shown. *, p<0.05, * *, p<0.01, * * *, p<0.001. (B) Inhibition of the pGL3-Ep and pGL3-Ap luciferase reporter genes by ZHX2 in HepG2 cells. Data (mean±SEM) of four experiments are shown. * *, p<0.01. (C) Diagram of the Cyclin E and Cyclin A promoters, extending to -402 and -505, respectively, including the NF-Y site in the cyclin A promoter and location of the primers (solid or dotted lines under promoters) used for ChIP analysis. (D) ChIP analysis of DNA from HepG2 cells transfected with ZHX2-HA. PCR amplification of HA-immunoprecipitated DNA using the primers shown in (C) shows ZHX2 binding to Cyclin A and Cyclin E promoters but not Cyclin D1. One of three independent experiments is shown. (E) Colony formation of QSG7701 and SMMC7721 cells transfected with shRNAs against ZHX2 (pS1674 or pS2360) and siRNAs agaist Cyclin A (sicycA) or Cyclin E (sicycE). One representative plate of each group is shown on the left; results from three independent experiments are shown in the right. (mean±SD) * * *, p<0.001. (F) Proliferation of QSG7701 and SMMC7721 cells that were co-transfected with ZHX2 shRNAs along with siRNAs for Cyclin A, Cyclin E or Cyclin D1. Cells were measured using CCK-8 48h after cotransfection. Mean±SD of three experiments is shown. ; * * *, p<0.001.

Figure 3. ZHX2 binds to and represses activity of the Cyclin A and Cyclin E promoters

(A) Real-time RT-PCR analysis of ZHX2, Cyclin A and Cyclin E mRNA levels in HepG2 cells transfected with pcZHX2 or pcDNA3.0 or SMMC7721 cells transfected with shRNAs. Data (mean±SD) of three experiments are shown. *, p<0.05, * *, p<0.01, * * *, p<0.001. (B) Inhibition of the pGL3-Ep and pGL3-Ap luciferase reporter genes by ZHX2 in HepG2 cells. Data (mean±SEM) of four experiments are shown. * *, p<0.01. (C) Diagram of the Cyclin E and Cyclin A promoters, extending to -402 and -505, respectively, including the NF-Y site in the cyclin A promoter and location of the primers (solid or dotted lines under promoters) used for ChIP analysis. (D) ChIP analysis of DNA from HepG2 cells transfected with ZHX2-HA. PCR amplification of HA-immunoprecipitated DNA using the primers shown in (C) shows ZHX2 binding to Cyclin A and Cyclin E promoters but not Cyclin D1. One of three independent experiments is shown. (E) Colony formation of QSG7701 and SMMC7721 cells transfected with shRNAs against ZHX2 (pS1674 or pS2360) and siRNAs agaist Cyclin A (sicycA) or Cyclin E (sicycE). One representative plate of each group is shown on the left; results from three independent experiments are shown in the right. (mean±SD) * * *, p<0.001. (F) Proliferation of QSG7701 and SMMC7721 cells that were co-transfected with ZHX2 shRNAs along with siRNAs for Cyclin A, Cyclin E or Cyclin D1. Cells were measured using CCK-8 48h after cotransfection. Mean±SD of three experiments is shown. ; * * *, p<0.001.

Figure 3. ZHX2 binds to and represses activity of the Cyclin A and Cyclin E promoters

(A) Real-time RT-PCR analysis of ZHX2, Cyclin A and Cyclin E mRNA levels in HepG2 cells transfected with pcZHX2 or pcDNA3.0 or SMMC7721 cells transfected with shRNAs. Data (mean±SD) of three experiments are shown. *, p<0.05, * *, p<0.01, * * *, p<0.001. (B) Inhibition of the pGL3-Ep and pGL3-Ap luciferase reporter genes by ZHX2 in HepG2 cells. Data (mean±SEM) of four experiments are shown. * *, p<0.01. (C) Diagram of the Cyclin E and Cyclin A promoters, extending to -402 and -505, respectively, including the NF-Y site in the cyclin A promoter and location of the primers (solid or dotted lines under promoters) used for ChIP analysis. (D) ChIP analysis of DNA from HepG2 cells transfected with ZHX2-HA. PCR amplification of HA-immunoprecipitated DNA using the primers shown in (C) shows ZHX2 binding to Cyclin A and Cyclin E promoters but not Cyclin D1. One of three independent experiments is shown. (E) Colony formation of QSG7701 and SMMC7721 cells transfected with shRNAs against ZHX2 (pS1674 or pS2360) and siRNAs agaist Cyclin A (sicycA) or Cyclin E (sicycE). One representative plate of each group is shown on the left; results from three independent experiments are shown in the right. (mean±SD) * * *, p<0.001. (F) Proliferation of QSG7701 and SMMC7721 cells that were co-transfected with ZHX2 shRNAs along with siRNAs for Cyclin A, Cyclin E or Cyclin D1. Cells were measured using CCK-8 48h after cotransfection. Mean±SD of three experiments is shown. ; * * *, p<0.001.

Figure 3. ZHX2 binds to and represses activity of the Cyclin A and Cyclin E promoters

(A) Real-time RT-PCR analysis of ZHX2, Cyclin A and Cyclin E mRNA levels in HepG2 cells transfected with pcZHX2 or pcDNA3.0 or SMMC7721 cells transfected with shRNAs. Data (mean±SD) of three experiments are shown. *, p<0.05, * *, p<0.01, * * *, p<0.001. (B) Inhibition of the pGL3-Ep and pGL3-Ap luciferase reporter genes by ZHX2 in HepG2 cells. Data (mean±SEM) of four experiments are shown. * *, p<0.01. (C) Diagram of the Cyclin E and Cyclin A promoters, extending to -402 and -505, respectively, including the NF-Y site in the cyclin A promoter and location of the primers (solid or dotted lines under promoters) used for ChIP analysis. (D) ChIP analysis of DNA from HepG2 cells transfected with ZHX2-HA. PCR amplification of HA-immunoprecipitated DNA using the primers shown in (C) shows ZHX2 binding to Cyclin A and Cyclin E promoters but not Cyclin D1. One of three independent experiments is shown. (E) Colony formation of QSG7701 and SMMC7721 cells transfected with shRNAs against ZHX2 (pS1674 or pS2360) and siRNAs agaist Cyclin A (sicycA) or Cyclin E (sicycE). One representative plate of each group is shown on the left; results from three independent experiments are shown in the right. (mean±SD) * * *, p<0.001. (F) Proliferation of QSG7701 and SMMC7721 cells that were co-transfected with ZHX2 shRNAs along with siRNAs for Cyclin A, Cyclin E or Cyclin D1. Cells were measured using CCK-8 48h after cotransfection. Mean±SD of three experiments is shown. ; * * *, p<0.001.

Figure 3. ZHX2 binds to and represses activity of the Cyclin A and Cyclin E promoters

(A) Real-time RT-PCR analysis of ZHX2, Cyclin A and Cyclin E mRNA levels in HepG2 cells transfected with pcZHX2 or pcDNA3.0 or SMMC7721 cells transfected with shRNAs. Data (mean±SD) of three experiments are shown. *, p<0.05, * *, p<0.01, * * *, p<0.001. (B) Inhibition of the pGL3-Ep and pGL3-Ap luciferase reporter genes by ZHX2 in HepG2 cells. Data (mean±SEM) of four experiments are shown. * *, p<0.01. (C) Diagram of the Cyclin E and Cyclin A promoters, extending to -402 and -505, respectively, including the NF-Y site in the cyclin A promoter and location of the primers (solid or dotted lines under promoters) used for ChIP analysis. (D) ChIP analysis of DNA from HepG2 cells transfected with ZHX2-HA. PCR amplification of HA-immunoprecipitated DNA using the primers shown in (C) shows ZHX2 binding to Cyclin A and Cyclin E promoters but not Cyclin D1. One of three independent experiments is shown. (E) Colony formation of QSG7701 and SMMC7721 cells transfected with shRNAs against ZHX2 (pS1674 or pS2360) and siRNAs agaist Cyclin A (sicycA) or Cyclin E (sicycE). One representative plate of each group is shown on the left; results from three independent experiments are shown in the right. (mean±SD) * * *, p<0.001. (F) Proliferation of QSG7701 and SMMC7721 cells that were co-transfected with ZHX2 shRNAs along with siRNAs for Cyclin A, Cyclin E or Cyclin D1. Cells were measured using CCK-8 48h after cotransfection. Mean±SD of three experiments is shown. ; * * *, p<0.001.

Figure 4. ZHX2 expression in HCC and correlation with clinical parameters

(A) Immunohistochemical staining of ZHX2 in HCC sections (left and right panels) and non-tumor liver sections (middle panel). T=tumor and P=adjacent non-tumor. Original magnification 200×. (B) Western blot analysis of ZHX2 levels in nuclear extracts of adjacent non-tumor (P) and tumor (T) samples from patients with HCC. Histone H2A.X was used as a control. Statistical data is shown at right. * * *, p<0.001. (C) Immunohistochemical staining of ZHX2 (left), Cyclin A (middle) and Cyclin E (right) in adjacent sections of a cancer biopsy from one patients. Original magnification 200×. (D) Immunohistochemical staining of ZHX2 and Ki-67 (marker of cell proliferation) in continuous biopsies (tumor sections and adjacent nontumor sections). (E) Statistical analysis of ZHX2 nuclear expression in poor, moderate or highly differentiated HCC samples. The immunoreactive score is shown as median±SD. * *, p<0.01. (F) Nuclear ZHX2 expression correlates with overall survival (left), Ki-67 (middle), and intratumoral microvascular density (right).

Figure 4. ZHX2 expression in HCC and correlation with clinical parameters

(A) Immunohistochemical staining of ZHX2 in HCC sections (left and right panels) and non-tumor liver sections (middle panel). T=tumor and P=adjacent non-tumor. Original magnification 200×. (B) Western blot analysis of ZHX2 levels in nuclear extracts of adjacent non-tumor (P) and tumor (T) samples from patients with HCC. Histone H2A.X was used as a control. Statistical data is shown at right. * * *, p<0.001. (C) Immunohistochemical staining of ZHX2 (left), Cyclin A (middle) and Cyclin E (right) in adjacent sections of a cancer biopsy from one patients. Original magnification 200×. (D) Immunohistochemical staining of ZHX2 and Ki-67 (marker of cell proliferation) in continuous biopsies (tumor sections and adjacent nontumor sections). (E) Statistical analysis of ZHX2 nuclear expression in poor, moderate or highly differentiated HCC samples. The immunoreactive score is shown as median±SD. * *, p<0.01. (F) Nuclear ZHX2 expression correlates with overall survival (left), Ki-67 (middle), and intratumoral microvascular density (right).

Figure 4. ZHX2 expression in HCC and correlation with clinical parameters

(A) Immunohistochemical staining of ZHX2 in HCC sections (left and right panels) and non-tumor liver sections (middle panel). T=tumor and P=adjacent non-tumor. Original magnification 200×. (B) Western blot analysis of ZHX2 levels in nuclear extracts of adjacent non-tumor (P) and tumor (T) samples from patients with HCC. Histone H2A.X was used as a control. Statistical data is shown at right. * * *, p<0.001. (C) Immunohistochemical staining of ZHX2 (left), Cyclin A (middle) and Cyclin E (right) in adjacent sections of a cancer biopsy from one patients. Original magnification 200×. (D) Immunohistochemical staining of ZHX2 and Ki-67 (marker of cell proliferation) in continuous biopsies (tumor sections and adjacent nontumor sections). (E) Statistical analysis of ZHX2 nuclear expression in poor, moderate or highly differentiated HCC samples. The immunoreactive score is shown as median±SD. * *, p<0.01. (F) Nuclear ZHX2 expression correlates with overall survival (left), Ki-67 (middle), and intratumoral microvascular density (right).

Figure 4. ZHX2 expression in HCC and correlation with clinical parameters

(A) Immunohistochemical staining of ZHX2 in HCC sections (left and right panels) and non-tumor liver sections (middle panel). T=tumor and P=adjacent non-tumor. Original magnification 200×. (B) Western blot analysis of ZHX2 levels in nuclear extracts of adjacent non-tumor (P) and tumor (T) samples from patients with HCC. Histone H2A.X was used as a control. Statistical data is shown at right. * * *, p<0.001. (C) Immunohistochemical staining of ZHX2 (left), Cyclin A (middle) and Cyclin E (right) in adjacent sections of a cancer biopsy from one patients. Original magnification 200×. (D) Immunohistochemical staining of ZHX2 and Ki-67 (marker of cell proliferation) in continuous biopsies (tumor sections and adjacent nontumor sections). (E) Statistical analysis of ZHX2 nuclear expression in poor, moderate or highly differentiated HCC samples. The immunoreactive score is shown as median±SD. * *, p<0.01. (F) Nuclear ZHX2 expression correlates with overall survival (left), Ki-67 (middle), and intratumoral microvascular density (right).

Figure 4. ZHX2 expression in HCC and correlation with clinical parameters

(A) Immunohistochemical staining of ZHX2 in HCC sections (left and right panels) and non-tumor liver sections (middle panel). T=tumor and P=adjacent non-tumor. Original magnification 200×. (B) Western blot analysis of ZHX2 levels in nuclear extracts of adjacent non-tumor (P) and tumor (T) samples from patients with HCC. Histone H2A.X was used as a control. Statistical data is shown at right. * * *, p<0.001. (C) Immunohistochemical staining of ZHX2 (left), Cyclin A (middle) and Cyclin E (right) in adjacent sections of a cancer biopsy from one patients. Original magnification 200×. (D) Immunohistochemical staining of ZHX2 and Ki-67 (marker of cell proliferation) in continuous biopsies (tumor sections and adjacent nontumor sections). (E) Statistical analysis of ZHX2 nuclear expression in poor, moderate or highly differentiated HCC samples. The immunoreactive score is shown as median±SD. * *, p<0.01. (F) Nuclear ZHX2 expression correlates with overall survival (left), Ki-67 (middle), and intratumoral microvascular density (right).

Figure 4. ZHX2 expression in HCC and correlation with clinical parameters

(A) Immunohistochemical staining of ZHX2 in HCC sections (left and right panels) and non-tumor liver sections (middle panel). T=tumor and P=adjacent non-tumor. Original magnification 200×. (B) Western blot analysis of ZHX2 levels in nuclear extracts of adjacent non-tumor (P) and tumor (T) samples from patients with HCC. Histone H2A.X was used as a control. Statistical data is shown at right. * * *, p<0.001. (C) Immunohistochemical staining of ZHX2 (left), Cyclin A (middle) and Cyclin E (right) in adjacent sections of a cancer biopsy from one patients. Original magnification 200×. (D) Immunohistochemical staining of ZHX2 and Ki-67 (marker of cell proliferation) in continuous biopsies (tumor sections and adjacent nontumor sections). (E) Statistical analysis of ZHX2 nuclear expression in poor, moderate or highly differentiated HCC samples. The immunoreactive score is shown as median±SD. * *, p<0.01. (F) Nuclear ZHX2 expression correlates with overall survival (left), Ki-67 (middle), and intratumoral microvascular density (right).

Figure 5. Nuclear ZHX2 localization is essential for growth inhibition in vitro and in vivo

CHO and 293 cells were transiently transfected with pZHX2(242-446), pZHX2(242-439), full-length pZHX2 and pEGFP-N1. (A) ZHX2 localization was determined by fluorescence microscopy of EGFP. DAP (blue) was used to stain nuclei. Original magnification 200×. (B) Western blot analysis of EGFP and EGFP fusion proteins in cytoplasm and nuclei. (C) Proliferation of HepG2 for four days after transfection with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. * * *, p<0.001. (D) Activity of the pGL3-Ep and pGL3Ap in HepG2 cells co-transfected with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. Data shown is mean±SD of three independent experiments. * *, p<0.01 * * *, p<0.001. (E and F) Tumors of HepG2.2.15 cells grown in nude mice were injected with pZHX2(242-439), pZHX2(242-446) or pEGFP-N1. (E) Weights of tumors were determined after sacrifice. (mean±SD ; n=6) *, p<0.05. (F) Tumor volume calculated every other day over 12 days (mean±SD ; n=6). * * *, p<0.001.

Figure 5. Nuclear ZHX2 localization is essential for growth inhibition in vitro and in vivo

CHO and 293 cells were transiently transfected with pZHX2(242-446), pZHX2(242-439), full-length pZHX2 and pEGFP-N1. (A) ZHX2 localization was determined by fluorescence microscopy of EGFP. DAP (blue) was used to stain nuclei. Original magnification 200×. (B) Western blot analysis of EGFP and EGFP fusion proteins in cytoplasm and nuclei. (C) Proliferation of HepG2 for four days after transfection with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. * * *, p<0.001. (D) Activity of the pGL3-Ep and pGL3Ap in HepG2 cells co-transfected with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. Data shown is mean±SD of three independent experiments. * *, p<0.01 * * *, p<0.001. (E and F) Tumors of HepG2.2.15 cells grown in nude mice were injected with pZHX2(242-439), pZHX2(242-446) or pEGFP-N1. (E) Weights of tumors were determined after sacrifice. (mean±SD ; n=6) *, p<0.05. (F) Tumor volume calculated every other day over 12 days (mean±SD ; n=6). * * *, p<0.001.

Figure 5. Nuclear ZHX2 localization is essential for growth inhibition in vitro and in vivo

CHO and 293 cells were transiently transfected with pZHX2(242-446), pZHX2(242-439), full-length pZHX2 and pEGFP-N1. (A) ZHX2 localization was determined by fluorescence microscopy of EGFP. DAP (blue) was used to stain nuclei. Original magnification 200×. (B) Western blot analysis of EGFP and EGFP fusion proteins in cytoplasm and nuclei. (C) Proliferation of HepG2 for four days after transfection with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. * * *, p<0.001. (D) Activity of the pGL3-Ep and pGL3Ap in HepG2 cells co-transfected with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. Data shown is mean±SD of three independent experiments. * *, p<0.01 * * *, p<0.001. (E and F) Tumors of HepG2.2.15 cells grown in nude mice were injected with pZHX2(242-439), pZHX2(242-446) or pEGFP-N1. (E) Weights of tumors were determined after sacrifice. (mean±SD ; n=6) *, p<0.05. (F) Tumor volume calculated every other day over 12 days (mean±SD ; n=6). * * *, p<0.001.

Figure 5. Nuclear ZHX2 localization is essential for growth inhibition in vitro and in vivo

CHO and 293 cells were transiently transfected with pZHX2(242-446), pZHX2(242-439), full-length pZHX2 and pEGFP-N1. (A) ZHX2 localization was determined by fluorescence microscopy of EGFP. DAP (blue) was used to stain nuclei. Original magnification 200×. (B) Western blot analysis of EGFP and EGFP fusion proteins in cytoplasm and nuclei. (C) Proliferation of HepG2 for four days after transfection with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. * * *, p<0.001. (D) Activity of the pGL3-Ep and pGL3Ap in HepG2 cells co-transfected with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. Data shown is mean±SD of three independent experiments. * *, p<0.01 * * *, p<0.001. (E and F) Tumors of HepG2.2.15 cells grown in nude mice were injected with pZHX2(242-439), pZHX2(242-446) or pEGFP-N1. (E) Weights of tumors were determined after sacrifice. (mean±SD ; n=6) *, p<0.05. (F) Tumor volume calculated every other day over 12 days (mean±SD ; n=6). * * *, p<0.001.

Figure 5. Nuclear ZHX2 localization is essential for growth inhibition in vitro and in vivo

CHO and 293 cells were transiently transfected with pZHX2(242-446), pZHX2(242-439), full-length pZHX2 and pEGFP-N1. (A) ZHX2 localization was determined by fluorescence microscopy of EGFP. DAP (blue) was used to stain nuclei. Original magnification 200×. (B) Western blot analysis of EGFP and EGFP fusion proteins in cytoplasm and nuclei. (C) Proliferation of HepG2 for four days after transfection with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. * * *, p<0.001. (D) Activity of the pGL3-Ep and pGL3Ap in HepG2 cells co-transfected with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. Data shown is mean±SD of three independent experiments. * *, p<0.01 * * *, p<0.001. (E and F) Tumors of HepG2.2.15 cells grown in nude mice were injected with pZHX2(242-439), pZHX2(242-446) or pEGFP-N1. (E) Weights of tumors were determined after sacrifice. (mean±SD ; n=6) *, p<0.05. (F) Tumor volume calculated every other day over 12 days (mean±SD ; n=6). * * *, p<0.001.

Figure 5. Nuclear ZHX2 localization is essential for growth inhibition in vitro and in vivo

CHO and 293 cells were transiently transfected with pZHX2(242-446), pZHX2(242-439), full-length pZHX2 and pEGFP-N1. (A) ZHX2 localization was determined by fluorescence microscopy of EGFP. DAP (blue) was used to stain nuclei. Original magnification 200×. (B) Western blot analysis of EGFP and EGFP fusion proteins in cytoplasm and nuclei. (C) Proliferation of HepG2 for four days after transfection with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. * * *, p<0.001. (D) Activity of the pGL3-Ep and pGL3Ap in HepG2 cells co-transfected with pZHX2(242-439), pZHX2(242-446) and pEGFP-N1. Data shown is mean±SD of three independent experiments. * *, p<0.01 * * *, p<0.001. (E and F) Tumors of HepG2.2.15 cells grown in nude mice were injected with pZHX2(242-439), pZHX2(242-446) or pEGFP-N1. (E) Weights of tumors were determined after sacrifice. (mean±SD ; n=6) *, p<0.05. (F) Tumor volume calculated every other day over 12 days (mean±SD ; n=6). * * *, p<0.001.