Journal: Biomedical Journal

Article Title: High expression of embryonic stem cell marker SSEA3 confers poor prognosis and promotes epithelial mesenchymal transition in hepatocellular carcinoma

doi: 10.1016/j.bj.2023.100612

Figure Lengend Snippet: SSEA3Cer-enhanced hepatocellular carcinoma (HCC) cancer cell migration and invasion were inhibited by ZEB1 silencing. (A) Effect of SSEA3Cer on expression level of transcription factors regulating migration and invasion related genes. HA22T, HA59T, and Hep3B cells were incubated with glucosylceramide (GlcCer) (Ctrl, left panel), 30 μM SSEA3Cer (middle panel) for 24 h. Total RNA was extracted for quantitation of SNAIL, TWIST1, and ZEB1 by quantitative real-time PCR. The mRNA levels were normalized to the level of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH and compared to the control cells. (B) HA22T, HA59T, and Hep3B cells were incubated with GlcCer (Ctrl, left panel), 30 μM SSEA3Cer (middle panel) or SSEA3Cer (30 μM)/SSEA3 Ab (5 μg) (right panel) for 24 h. Total RNA was extracted for quantitation of ZEB1 by quantitative real-time PCR. The mRNA levels were normalized to the level of GAPDH and compared to the control cells. (C–E) Boyden chamber assay was employed to examine the migration and invasion ability of HA22T, HA59T, and Hep3B cells. The cells were transfected with control siRNA (si-Ctrl) or ZEB1 siRNA (si-ZEB1) for 24 h. Then, the migration (C), invasion (D), and quantitative real-time PCR (E) assays were performed in the cells incubated with GlcCer or 30 μM SSEA3Cer for 24 h. Total RNA was extracted for quantitation of CDH1, CDH2, VIM, FN, MMP2, and ZEB1 by quantitative real-time PCR. The mRNA levels were normalized to the level of GAPDH and compared to the siCtrl/GlcCer cells. The data were presented as mean ± SD of triplicate determination. The results were from three independent experiments ( ∗ p < 0.05, One-way ANOVA test).

Article Snippet: Primer pairs for CDH1 (HP207683), CDH2 (HP205580), VIM (HP206907), FN (HP234005), MMP2 (HP207826), SNAIL (SNAI1, HP209016), TWIST1 (HP200446), ZEB1 (HP215380) and GAPDH (HP205798) were purchased from OriGene (Rockville, MD, USA).

Techniques: Migration, Expressing, Incubation, Quantitation Assay, Real-time Polymerase Chain Reaction, Control, Boyden Chamber Assay, Transfection

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-108

Figure Lengend Snippet: 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.

Article Snippet: The following antibodies were used: mouse anti-human HIF-1α monoclonal antibody (BD Clontech, USA), mouse anti-human E-cadherin monoclonal antibody, mouse anti-human N-cadherin monoclonal antibody, mouse anti-human Vimentin monoclonal antibody, rabbit anti-human Twist polyclonal antibody and rabbit anti-human SNAI1 polyclonal antibody (Santa Cruz Biotech, USA).

Techniques: Over Expression, Immunohistochemistry, Expressing

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-108

Figure Lengend Snippet: 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 CoCl 2 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 CoCl 2 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 CoCl 2 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.

Article Snippet: The following antibodies were used: mouse anti-human HIF-1α monoclonal antibody (BD Clontech, USA), mouse anti-human E-cadherin monoclonal antibody, mouse anti-human N-cadherin monoclonal antibody, mouse anti-human Vimentin monoclonal antibody, rabbit anti-human Twist polyclonal antibody and rabbit anti-human SNAI1 polyclonal antibody (Santa Cruz Biotech, USA).

Techniques: Expressing, Western Blot, Staining

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-108

Figure Lengend Snippet: 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 CoCl 2 -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 CoCl 2 -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 CoCl 2 -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.

Article Snippet: The following antibodies were used: mouse anti-human HIF-1α monoclonal antibody (BD Clontech, USA), mouse anti-human E-cadherin monoclonal antibody, mouse anti-human N-cadherin monoclonal antibody, mouse anti-human Vimentin monoclonal antibody, rabbit anti-human Twist polyclonal antibody and rabbit anti-human SNAI1 polyclonal antibody (Santa Cruz Biotech, USA).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Staining

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-108

Figure Lengend Snippet: 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.

Article Snippet: The following antibodies were used: mouse anti-human HIF-1α monoclonal antibody (BD Clontech, USA), mouse anti-human E-cadherin monoclonal antibody, mouse anti-human N-cadherin monoclonal antibody, mouse anti-human Vimentin monoclonal antibody, rabbit anti-human Twist polyclonal antibody and rabbit anti-human SNAI1 polyclonal antibody (Santa Cruz Biotech, USA).

Techniques: Luciferase, Construct, Transfection, Plasmid Preparation, Expressing, Activity Assay