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. 2017 Jun 24;36(1):86.
doi: 10.1186/s13046-017-0559-4.

HOXB7 accelerates the malignant progression of hepatocellular carcinoma by promoting stemness and epithelial-mesenchymal transition

Hong-Bo Huan  1 , Da-Peng Yang  1 , Xu-Dong Wen  1 , Xue-Jiao Chen  2 , Liang Zhang  1 , Li-Li Wu  1 , Ping Bie  1 , Feng Xia  3
Affiliations

HOXB7 accelerates the malignant progression of hepatocellular carcinoma by promoting stemness and epithelial-mesenchymal transition

Hong-Bo Huan et al. J Exp Clin Cancer Res. .

Abstract

Background: Homeobox B7 (HOXB7) has been identified associated with poor prognosis of hepatocellular carcinoma (HCC). However, the specific mechanism by which HOXB7 promotes the malignant progression of HCC remains to be determined.

Methods: Immunohistochemistry (IHC) was used to detect the expression level of HOXB7 in 77-paired HCC tissue samples, and the correlation between HOXB7 and HCC prognosis was assessed. The location of HOXB7 was confirmed by immunofluorescence. Cell Titer-Blue assay was used to assess the proliferation of hepatoma cells. The stem-like properties of hepatoma cells were analysed by sphere formation and clone formation assays. The effect of HOXB7 on expression of cancer stem cell markers was evaluated. Transwell and wound-healing assays were performed to estimate the invasion and migration abilities of hepatoma cells. A xenograft tumor model was established in nude mice to assess the role of HOXB7 in tumor growth. Bioluminescence imaging was used to survey the effect of HOXB7 on the metastatic ability of hepatoma cells in vivo.

Results: Higher expression of HOXB7 was detected in HCC tissues compared with noncancerous tissues and significantly associated with poor prognosis of HCC. In addition, HOXB7 knockdown suppressed the cell proliferation, clone formation, sphere formation, invasion and migration of hepatoma cells in vitro; conversely, these biological abilities of hepatoma cells were enhanced by HOXB7 overexpression. Moreover, the cancer stem cell markers EPCAM and NANOG were up-regulated by HOXB7. The role of HOXB7 in promoting tumor growth and metastasis was verified in vivo. Further investigation revealed that c-Myc and Slug expression was elevated by HOXB7 and the AKT pathway was activated.

Conclusion: Overexpression of HOXB7 was significantly correlated with poor prognosis of HCC. HOXB7 up-regulated c-Myc and Slug expression via the AKT pathway to promote the acquisition of stem-like properties and facilitate epithelial-mesenchymal transition of hepatoma cells, accelerating the malignant progression of HCC.

Keywords: Epithelial-mesenchymal transition; Hepatocellular carcinoma; Homeobox B7; Slug; Stemness; c-Myc.

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Conflict of interest statement

Ethics approval and consent to participate

All patients were informed with consent according to protocols approved by the Institutional Review Board of the Southwest Hospital, Third Military Medical University, and this study complied with by the ethical guidelines of the Helsinki Declaration. Moreover, the animal research was approved by the Animal Research Ethics Committee of Third Military Medical University, and complied with the Guidelines for Animal Experiments of Laboratory Animals.

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Not applicable

Competing interests

The authors declare that they have no competing interest.

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Figures

Fig. 1
Fig. 1
Up-regulation of HOXB7 in HCC tissues correlates with poor prognosis of HCC. a A representative image showing the expression level of HOXB7 in HCC and paired adjacent noncancerous tissues. HOXB7 was negative in adjacent noncancerous tissue and positive in HCC tissue. b IHC staining scores were used to define low and high expression of HOXB7 in HCC tissues (n = 77; P < 0.01). c Upper panel: protein level of HOXB7 in 10 randomly selected paired HCC tissues was determined by western blot. Lower panel: semi-quantitative analysis of the western blot results. GAPDH was used to normalize the expression level of HOXB7 (n = 10; P < 0.001). d Kaplan-Meier survival analysis of HCC patients. A high level of HOXB7 resulted in a shorter survival time in HCC patients (n = 37); a low level of HOXB7 was correlated with a longer survival time (n = 40; P < 0.001)
Fig. 2
Fig. 2
HOXB7 facilitated hepatoma cell proliferation by promoting stemness. a Protein level of HOXB7 in different hepatoma cell lines. b Efficiency of knockdown and overexpression HOXB7 in SMMC-7721 and HepG2 cells, respectively. c Location of HOXB7 in SMMC-7721 and HepG2 cells. d Cell Titer-Blue assay was used to examine the effect of HOXB7 on the proliferation of SMMC-7721 and HepG2 cells. HOXB7 promoted the proliferation of hepatoma cells (P < 0.05, respectively). e Analysis of clone formation in SMMC-7721 and HepG2 cells. HOXB7 promoted clone formation of hepatoma cells (P < 0.05, respectively). f Analysis of sphere formation in SMMC-7721 and HepG2 cells. HOXB7 accelerated sphere formation of hepatoma cells (P < 0.05, respectively). g, h Hepatoma stem cell markers EPCAM and NANOG expression level after HOXB7 knockdown and overexpression
Fig. 3
Fig. 3
HOXB7 promoted EMT to enhance the migration and invasion of hepatoma cells. a Knockdown of HOXB7 inhibited the migration and invasion of SMMC-7721 cells; cell counting showed that invasion and migration were evidently inhibited by knockdown of HOXB7 in SMMC-7721 cells (P < 0.001, respectively). b Overexpression of HOXB7 facilitated HepG2 cell migration and invasion (P < 0.001, respectively). All images were taken under × 100 magnification. c, d Wound healing assay was used to examine the effect of HOXB7 on SMMC-7721 and HepG2 cells migration. Both figures revealed that HOXB7 promoted hepatoma cells migration. e HOXB7 reduced the expression of E-cadherin and up-regulated the expression of N-cadherin, Vimentin, α-SMA, MMP2 and MMP9 in SMMC-7721 and HepG2 cells, respectively. f Changes of cell morphology after overexpression of HOXB7
Fig. 4
Fig. 4
Overexpression of HOXB7 facilitated tumor growth in vivo. a SMMC-7721/scramble, SMMC-7721/shHOXB7, HepG2/vector and HepG2/HOXB7 cells were subcutaneously injected in the hind limbs of nude mice (n = 6/group; 1 × 105 cells). b, c Tumor weight and volume were measured in each group (P < 0.05, respectively). d, e Representative histopathology of xenograft tumors. The tumor sections were subjected to IHC staining using antibodies against HOXB7 and Ki-67
Fig. 5
Fig. 5
Effect of HOXB7 on metastasis of hepatoma cells in vivo. a, e Representative images of metastasis to the lung in the SMMC-7721/scramble, SMMC-7721/shHOXB7, HepG2/vector and HepG2/HOXB7 groups (n = 6 per group). b, f Quantification of fluorescence from metastatic tumors (P < 0.01, respectively). c, g H&E staining of metastatic tumors in lung tissues. d, h Kaplan-Meier survival analysis of mice in different groups
Fig. 6
Fig. 6
HOXB7 up-regulated c-Myc and Slug expression via the AKT pathway to promote HCC progression. a, b RT-PCR was used to investigate the effect of HOXB7 on stemness and metastasis related genes such as Sox2, Sox9, c-Myc, Oct4, Slug and Twist1 in SMMC-7721 and HepG2 cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001). c, d The expression of AKT, p-AKT, ERK, p-ERK, p-38, p-p38, SMAD3 and p-SMAD3 in hepatoma cells were analysed by western blot. e, f HOXB7 up-regulated EPCAM, NANOG, Slug, c-Myc and promoted phosphorylation of AKT. g Pharmacologically inhibiting AKT depressed the effect of HOXB7 on c-Myc and Slug expression
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References

    1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108. doi: 10.3322/caac.21262. - DOI - PubMed
    1. Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol. 2006;45:529–38. doi: 10.1016/j.jhep.2006.05.013. - DOI - PubMed
    1. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379:1245–55. doi: 10.1016/S0140-6736(11)61347-0. - DOI - PubMed
    1. Tschopp P, Duboule D. A genetic approach to the transcriptional regulation of Hox gene clusters. Annu Rev Genet. 2011;45:145–66. doi: 10.1146/annurev-genet-102209-163429. - DOI - PubMed
    1. Shah N, Sukumar S. The Hox genes and their roles in oncogenesis. Nat Rev Cancer. 2010;10:361–71. doi: 10.1038/nrc2826. - DOI - PubMed

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