Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr;52(4):1235-1245.
doi: 10.3892/ijo.2018.4292. Epub 2018 Feb 28.

H2A.Z regulates tumorigenesis, metastasis and sensitivity to cisplatin in intrahepatic cholangiocarcinoma

Beng Yang  1 , Rongliang Tong  1 , Hua Liu  1 , Jingbang Wu  1 , Diyu Chen  1 , Zhengze Xue  2 , Chaofeng Ding  1 , Lin Zhou  3 , Haiyang Xie  3 , Jian Wu  1 , Shusen Zheng  1
Affiliations

H2A.Z regulates tumorigenesis, metastasis and sensitivity to cisplatin in intrahepatic cholangiocarcinoma

Beng Yang et al. Int J Oncol. 2018 Apr.

Abstract

Intrahepatic cholangiocarcinoma (ICC) is a fatal, malignant tumor of the liver; effective diagnostic biomarkers and therapeutic targets for ICC have not been identified yet. High expression of H2A histone family member Z (H2A.Z) is a high-risk factor for poor prognosis in patients with breast cancer and primary hepatocellular cancer. However, the significance of H2A.Z and its expression in ICC remains unknown. The present study demonstrated that H2A.Z is overexpressed in ICC and expression of H2A.Z correlated with poor prognosis in patients with ICC. H2A.Z regulated cell proliferation in vitro and in vivo via H2A.Z/S-phase kinase-associated protein 2/p27/p21 signaling. Inhibition of H2A.Z reduced cell proliferation and induced apoptosis in ICC. In addition, downregulation of H2AZ reduced tumor metastasis by repressing epithelial-mesenchymal transition and enhanced the antitumor effects of cisplatin in the treatment of ICC. Overall, H2A.Z promoted cell proliferation and epithelial-mesenchymal transition in ICC, suggesting that H2A.Z may be a novel biomarker and therapeutic target for ICC.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
H2A.Z is highly expressed in ICC tissues and associated with poorer prognosis of ICC patients. (A) Representative images from immunohistochemical analysis of H2A.Z expression in paired ICC and normal tissue. (B) Total staining scores from the H2A.Z immunohistochemistry results in paired and peritumoral tissues. (C) H2A.Z protein expression levels were examined by western blotting in 8 pairs of ICC and normal peritumoral tissues. (D) Association between H2A.Z expression and overall survival in patients with ICC. **P<0.01. H2A.Z, H2A histone family member Z; ICC, intrahepatic cholangiocarcinoma; N, normal; T, tumor.
Figure 2
Figure 2
H2A.Z knockdown inhibits cell proliferation in ICC cells in vitro. (A) H2A.Z protein expression levels were examined by western blotting in various ICC cell lines (CCLP-1, HuCCT-1, RBE and HCCC-9810) and the normal HIBEC cell line. (B) H2A.Z knockdown stable cell lines were constructed in CCLP1 and HCCC-9810 cells by shRNA lentiviral transduction. (C) The efficacy of H2A.Z silencing by siRNA transfection in CCLP1 and HCCC-9810 cells was confirmed by western blotting. (D and E) Colony formation assay results for the H2A.Z knockdown (conducted both by shRNA and siRNA) or negative control CCLP1 and HCCC-9810 cells. (F and G) Cell Counting Kit-8 assay results for either siRNA or control-transfected CCLP1 and HCCC-9810 cells. Every experiment was repeated three times. **P<0.01 and ***P<0.001 compared with NC. H2A.Z, H2A histone family member Z; ICC, intrahepatic cholangiocarcinoma; HIBEC, human intrahepatic biliary epithelial cell; sh, short hairpin; si, small interfering; NC, negative control.
Figure 3
Figure 3
H2A.Z knockdown induces cell cycle arrest in ICC cells. (A) Cell proliferation was detected by EdU assay (red signal). Hoechst (blue signal) was used to counterstain the cell nuclei. Representative images and quantification are shown. (B and C) The effect of H2A.Z knockdown on cell cycle arrest was examined by flow cytometry. (D and E) CCLP-1 and HCCC-9810 cells were transfected with H2A.Z shRNA by lentivirus. The cell lysates were used to perform western blot analysis with the indicated antibodies and β-actin was used as a loading control. Every experiment was repeated three times. *P<0.05 and, **P<0.01 compared with NC. H2A.Z, H2A histone family member Z; ICC, intrahepatic cholangiocarcinoma; EdU, ethynyl deoxyuridine; sh, short hairpin; NC, negative control; Skp2, S-phase kinase-associated protein 2; p27, cyclin-dependent kinase inhibitor 1B; p21, cyclin-dependent kinase inhibitor 1A; CDK, cyclin-dependent kinase.
Figure 4
Figure 4
H2A.Z knockdown induces cell apoptosis in ICC cells. (A) The effect of H2A.Z knockdown on cell apoptosis was examined by flow cytometry, and the % of apoptotic cells was calculated in each experimental group. (B and C) CCLP-1 and HCCC-9810 cells were transfected with H2A.Z shRNA. The lysates were used to perform western blot analysis with the indicated antibodies and β-actin was used as a loading control. Every experiment was repeated three times. **P<0.01 compared with NC. H2A histone family member Z; ICC, intrahepatic cholangiocarcinoma; sh, short hairpin; NC, negative control; Bcl-2, B-cell lymphoma 2; Bak, Bcl-2 homologous antagonist/killer; cas, caspase; cad, cadherin.
Figure 5
Figure 5
H2A.Z knockdown inhibits migration and invasion in ICC cells. (A and B) H2A.Z knockdown significantly suppressed migration and invasion compared with control cells, as detected by Transwell assays. (C) Similar effects were observed in cell motility by wound healing assays. Every experiment was repeated 3 times. *P<0.05 and, **P<0.01 compared with NC. H2A histone family member Z; ICC, intrahepatic cholangiocarcinoma; sh, short hairpin; NC, negative control.
Figure 6
Figure 6
H2A.Z knockdown inhibits tumor growth and metastasis in vivo. (A) Photographic images of the xenograft tumors from the two experimental groups (knockdown and control). Tumor weight and volumes were measured. (B) Representative images from immunohistochemistry analysis of tumor sections for p21, Ki67 and H2A.Z protein expression. (C) Representative images of the lungs and of H&E-stained lung sections from mice that underwent experimental metastasis assay. Metastatic tumors in lungs are marked with arrows. The numbers of metastases per lung were calculated. (D) Apoptotic cells (brown staining) in sections from the xenograft tumors were detected by TUNEL assay in the control and the H2AZ knockdown groups. *P<0.05 and, **P<0.01 compared with NC. H2A histone family member Z; p21, cyclin-dependent kinase inhibitor 1A; H&E, hematoxylin and eosin; NC, negative control; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.
Figure 7
Figure 7
H2AZ knockdown strengthens the effect of cisplatin treatment in ICC cells. CCLP-1 cells were treated with shH2A.Z lentivirus. After 48 h, 4 µg/ml cisplatin was added in the medium and cells were incubated for another 24 h. (A) Cell morphology was captured by microscopy. (B) Apoptosis was examined by flow cytometry. (C) Cell viability was measured by Cell Counting Kit-8 assay. (D) Protein expression levels of apoptosis-related proteins were evaluated by western blotting. Every experiment was repeated three times. *P<0.05, **P<0.01 and ***P<0.001 compared with NC. H2A histone family member Z; ICC, intrahepatic cholangiocarcinoma; sh, short hairpin; NC, negative control; Cis, cisplatin; Bak, Bcl-2 homologous antagonist/killer; cas, caspase.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. McGlynn KA, Tarone RE, El-Serag HB. A comparison of trends in the incidence of hepatocellular carcinoma and intrahepatic cholangiocarcinoma in the United States. Cancer Epidemiol Biomarkers Prev. 2006;15:1198–1203. doi: 10.1158/1055-9965.EPI-05-0811. - DOI - PubMed
    1. Patel T. Increasing incidence and mortality of primary intra-hepatic cholangiocarcinoma in the United States. Hepatology. 2001;33:1353–1357. doi: 10.1053/jhep.2001.25087. - DOI - PubMed
    1. Patel T. Worldwide trends in mortality from biliary tract malignancies. BMC Cancer. 2002;2:10. doi: 10.1186/1471-2407-2-10. - DOI - PMC - PubMed
    1. Sia D, Tovar V, Moeini A, Llovet JM. Intrahepatic cholangiocarcinoma: Pathogenesis and rationale for molecular therapies. Oncogene. 2013;32:4861–4870. doi: 10.1038/onc.2012.617. - DOI - PMC - PubMed
    1. West MH, Bonner WM. Histone 2A, a heteromorphous family of eight protein species. Biochemistry. 1980;19:3238–3245. doi: 10.1021/bi00555a022. - DOI - PubMed

MeSH terms