Hypoxia induces epithelial-mesenchymal transition via activation of SNAI1 by hypoxia-inducible factor -1α in hepatocellular carcinoma - PubMed (original) (raw)

doi: 10.1186/1471-2407-13-108.

Gang Huang, Xiaowu Li, Yujun Zhang, Yan Jiang, Junjie Shen, Jia Liu, Qingliang Wang, Jin Zhu, Xiaobin Feng, Jiahong Dong, Cheng Qian

Affiliations

Hypoxia induces epithelial-mesenchymal transition via activation of SNAI1 by hypoxia-inducible factor -1α in hepatocellular carcinoma

Lin Zhang et al. BMC Cancer. 2013.

Abstract

Background: High invasion and metastasis are the primary factors causing poor prognosis of patients with hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying these biological behaviors have not been completely elucidated. In this study, we investigate the molecular mechanism by which hypoxia promotes HCC invasion and metastasis through inducing epithelial-mesenchymal transition (EMT).

Methods: The expression of EMT markers was analyzed by immunohistochemistry. Effect of hypoxia on induction of EMT and ability of cell migration and invasion were performed. Luciferase reporter system was used for evaluation of Snail regulation by hypoxia-inducible factor -1α (HIF-1α).

Results: We found that overexpression of HIF-1α was observed in HCC liver tissues and was related to poor prognosis of HCC patients. HIF-1α expression profile was correlated with the expression levels of SNAI1, E-cadherin, N-cadherin and Vimentin. Hypoxia was able to induce EMT and enhance ability of invasion and migration in HCC cells. The same phenomena were also observed in CoCl2-treated cells. The shRNA-mediated HIF-1α suppression abrogated CoCl2-induced EMT and reduced ability of migration and invasion in HCC cells. Luciferase assay showed that HIF-1α transcriptional regulated the expression of SNAI1 based on two hypoxia response elements (HREs) in SNAI1 promoter.

Conclusions: We demonstrated that hypoxia-stabilized HIF1α promoted EMT through increasing SNAI1 transcription in HCC cells. This data provided a potential therapeutic target for HCC treatment.

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Figures

Figure 1

Figure 1

Overexpression of HIF-1α in HCC is correlated with the level of SNAI1 and EMT markers and predicts poor prognosis. Immunohistochemistry was performed for determining the expression profile of various proteins, including HIF-1α (A, F), SNAI1 (B, G), E-cadherin (C, H), N-cadherin (D, I) and Vimentin (E, J), on HIF-1α+ (A-E) and HIF-1α- (F-J) HCC samples (A, D, F, I: ×200; B, C, E, G, H, J: ×400). (K) Disease-free survival after surgery was compared between HIF-1α positive (n = 43) and HIF-1α negative (n = 23) HCC patients.

Figure 2

Figure 2

Hypoxia induced EMT of HCC cells while reversion occurred under reoxygenation. (A) Morphological changes of hypoxia and reoxygenation-treated SMMC-7721 cells were recorded by light microscope (×200). (B) Immunohistochemical analysis of E-cadherin, N-cadherin and Vimentin expression was performed on both hypoxically and normoxically cultured SMMC-7721 (×200). (C) mRNA levels of E-cadherin, N-cadherin and Vimentin in SMMC-7721 was determined under the conditions of normoxia, hypoxia and reoxygenation by qPCR. GAPDH was used as endogenous reference and mRNA level of SMMC-7721 under normoxia was used as control. Data were shown as mean ± SD of three independent experiments. (D) Protein expression of E-cadherin, N-cadherin and Vimentin in SMMC-7721 cells undergoing normoxia, hypoxia and reoxygenation was determined by immunoblotting. β-actin was used as endogenous control. (E) The numbers of invasive and migrating SMMC-7721 cells undergoing normoxia, hypoxia and reoxygenation was calculated with crystal violet staining. The average numbers of ten random microscopic fields (×400) was recorded in each experiment. Data was shown as mean ± SD of three independent experiments. Representative images of each group were shown.

Figure 3

Figure 3

CoCl 2 -induced HIF-1α stabilization promotes SNAI1 expression, EMT and invasion capacity of HCC cells. (A) mRNA expression level of E-cadherin, N-cadherin, Vimentin, HIF-1α and SNAI1 was determined in SMMC-7721 cells with and without CoCl2 exposure (100 μM) by qPCR. GAPDH was used as endogenous control and mRNA level of untreated SMMC-7721cells was used as control. Data were shown as mean ± SD of three independent experiments. (B) Protein expression of E-cadherin, N-cadherin, Vimentin, HIF-1α and SNAI1 in SMMC-7721 cells with or without CoCl2 treatment (100 μM) was determined by immunoblotting. β-actin was used as endogenous reference. (C) The numbers of invasive and migrating SMMC-7721 cells with and without CoCl2 exposure (100 μM) was calculated with crystal violet staining. The average numbers of ten random microscopic fields (×400) was recorded in each experiment. Data was shown as mean ± SD of three independent experiments. Representative images of each group were shown.

Figure 4

Figure 4

HIF-1α silencing in HCC cells inhibits SNAI1-mediated EMT and invasion under CoCl 2 treatment. (A) mRNA levels of HIF-1α, SNAI1, E-cadherin, N-cadherin and Vimentin were determined in CoCl2-treated SMMC-7721 cells infected with Ad-scrambled or Ad-shHIF1α (10 MOI) by qRT-PCR. GAPDH was used as endogenous reference and mRNA level of untreated SMMC-7721cells was used as standard. Data were shown as mean ± SD of three independent experiments. (B) Protein levels of E-cadherin, N-cadherin and Vimentin in CoCl2-treated SMMC-7721 cells infected with Ad-scrambled or Ad-shHIF1a was determined by immunoblotting. β-actin was used as endogenous reference. (C) The numbers of invasive and migrating CoCl2-treated SMMC-7721 cells infected with Ad-scrambled or Ad-shHIF1α was calculated with crystal violet staining. The average numbers of ten random microscopic fields (×400) was recorded in each experiment. Data was shown as mean ± SD of three independent experiments. Representative images of each group were shown.

Figure 5

Figure 5

HIF-1α promotes transcription of SNAI1 under hypoxia condition. (A) Schematic diagram of SNAI1 promoter-luciferase construct was shown with the location of HRE. (HRE: hypoxia response element) (B) SMMC-7721 cells were transfected with pGL3-basic vector or a series of pGL3 vectors containing truncated SNAI1 promoters or promoters with mutated HRE (P1, P2, P3, P4, M1, M2 and MM) along with renilla luciferase expression vector. Luciferase assay was performed after 2 days. The firefly luciferase activity was normalized by renilla luciferase activity. Data were shown as mean ± SD of three independent experiments. (C) A model was shown for effect of HIF-1α on SNAI1-mediated EMT in HCC.

References

    1. Christofori G. New signals from the invasive front. Nature. 2006;441:444–450. doi: 10.1038/nature04872. - DOI - PubMed
    1. Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ, Teng SC, Wu KJ. Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol. 2008;10:295–305. doi: 10.1038/ncb1691. - DOI - PubMed
    1. Thompson EW, Williams ED. EMT and MET in carcinoma–clinical observations, regulatory pathways and new models. Clin Exp Metastasis. 2008;25:591–592. doi: 10.1007/s10585-008-9189-8. - DOI - PubMed
    1. Hill RP, Marie-Egyptienne DT, Hedley DW. Cancer stem cells, hypoxia and metastasis. Semin Radiat Oncol. 2009;19:106–111. doi: 10.1016/j.semradonc.2008.12.002. - DOI - PubMed
    1. Kudo-Saito C, Shirako H, Takeuchi T, Kawakami Y. Cancer Metastasis Is Accelerated through Immunosuppression during Snail-induced EMT of Cancer Cells. Cancer Cell. 2009;15:195–206. doi: 10.1016/j.ccr.2009.01.023. - DOI - PubMed

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