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.2004 Mar 22;90(6):1265-73.
doi: 10.1038/sj.bjc.6601685.

Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells

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Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells

A Miyoshi et al. Br J Cancer..

Abstract

Loss of E-cadherin (E-cad) triggers invasion, metastasis, and dedifferentiation in various epithelial carcinomas. Recently, it has been reported that two transcription factors, Snail and SIP1 (Smad interacting protein 1), directly repress transcription of the E-cad gene by binding E-box on E-cad promoter. Our aim is to solve the molecular mechanism of Snail and SIP1 in hepatocellular carcinoma (HCC). We first showed an inverse correlation between E-cad and Snail/SIP 1 expression among five HCC lines with different phenotypes. The result indicated that undifferentiated, but not differentiated type expressed Snail/SIP1. Then, we established transfectants stably expressing Snail and SIP1 in two differentiated cells with E-cad expression. Suppressed expression of E-cad, morphologic change into fibroblastoid feature, and remarkable acceleration of invasion activity were observed in the transfectants. In reverse transcription-polymerase chain reaction series of genes relating to motility and invasion, we demonstrated striking evidence that matrix metalloproteinase (MMP-1), MMP-2, MMP-7, and MT1-MMP expressions were strongly upregulated by Snail. On the other hand, MMP-1, MMP-2, and MT1-MMP expressions were enhanced by SIP1 transfection, however, the intensity was weaker than that in Snail transfection. In conclusion, Snail or SIP1 expression may be induced during HCC progression, where Snail/SIP1 directly represses E-cad gene transcription and activates cancer invasion via the upregulation of the MMP gene family.

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Figures

Figure 1
Figure 1
E-cadherin, Snail, and SIP1expressions were analysed and compared among five HCC cell lines by RT–PCR and the Western blot method. (A) RT–PCR: differentiated cell lines HepG2 and Huh-7 expressed E-cad but not Snail or SIP1. Undifferentiated cell lines HLF and Changliver expressed Snail and SIP1 but not E-cad. The undifferentiated cell line Hul-1 expressed Snail but not SIP1 or E-cad. GAPDH was used as an internal marker. (B) Western blot: E-cad protein was expressed in HepG2 and Huh-7 cell lines.β-actin protein levels were used to normalise the Western blot reactions.
Figure 2
Figure 2
(A) The human E-cad promoter fragment spanning −218 to +47 at the transcription start site, which contained three E-boxes elements was amplified by PCR using genomic DNA from HepG2. The promoter fragment was ligated upstream from the luciferase gene in the pGL3-basic plasmid (pGL3-E-cad). The repression effect on E-cad promoter activity by Snail and SIP1 in HepG2 and Huh-7 cell lines. Cells were cotransfected with 1 or 1.5 μg of pGL3-E-cad and various amounts of Snail or SIP1 expression plasmid. Luciferase activities were normalised byβ-galactosidase activities and shown by mean value±standard deviation of the triplicate measurements. (B, C) Snail repressed E-cad promoter activity. Both cell lines transfected with Snail expression plasmid showed luciferase activity at about one-third compared with control. (D) In the HepG2 cell line, the promoter activity was clearly repressed by SIP1 expression in a dose-dependent manner and the minimal activity was about 30% of the control activity. (E) In Huh-7 cells, the promoter activity in SIP1 transfection showed about 70% activity of control transfection.
Figure 3
Figure 3
Exogenous expression of Snail and SIP1 repressed E-cad mRNA and protein. HepG2 and Huh-7 cell lines were transfected with expression vector harbouring cDNA encoding HA epitope-tagged full-length mouse Snail (Snail-1, -2), FLAG-tagged full-length mouse SIP1 (SIP1-1, -2). Individual cell clones were isolated after selection by G-418. (A, B) E-cadherin, Snail, SIP1, and GAPDH expressions were determined by RT-PCR. GAPDH was used as an internal marker. (C, D) Western blot was carried out to examine E-cad, Snail, SIP1, andβ-actin protein expressions.β-actin protein levels were used to normalise the Western blot reactions.
Figure 4
Figure 4
Microscopic features of stable transfectant in HepG2 cells. (A) control; (B) Snail; (C) SIP1. Photographs were taken at a magnification of ×200. The morphologic alteration to fibroblastoid features was observed in Snail and SIP1 transfectants compared with control cells.
Figure 5
Figure 5
MTT proliferation assay was carried out using each transfectant cell. Cells (1×104) were plated on 96-well plates and after 24 h; MTT activities were measured in triplicate on days 1, 2, 3, and 4. The proliferation curves were illustrated by plotting the average of triplicated values calculated by optical density measurements at 570 nm in a 96-well plate reader. Relative proliferation values on days 2–4 were shown as ratio to OD570 nm on day 1. All transfectants in HepG2 and Huh-7 cells showed similar growth curves and there was no significant difference.
Figure 6
Figure 6
Invasion ability of each cell was analysed in the transwell invasion assay. Data are presented as mean value±standard deviation of the triplicate measurements. Snail and SIP1 transfectant cells showed significantly higher invasive properties compared with control cells (*P<0.05). In particular, Snail transfectants showed stronger invasive properties than SIP1 transfectants.
Figure 7
Figure 7
RT–PCR was performed using Snail and SIP1 transfectants in HepG2. (A) In candidate genes relating to cancer invasion, MMP-1, MMP-2, MMP-7 and MT1-MMP expressions were upregulated by Snail. Matrix metalloproteinase-1, MMP-2, and MT1-MMP were increased by SIP1 transfection. Genes for mesenchymal marker, vimentin, and fibronectin showed higher expression in Snail and SIP1 transfectants, compared with the control cells. (B) In quantitative RT–PCR analysis using Light-Cycler system, the expression amount of MMP genes was calculated by quantitative value with GAPDH. Relative expression rate for each MMP family was indicated as ratio to values with control. Mean value±standard deviation of the triplicate measurements was calculated and illustrated in the histogram of the right panel. Expression of MMP-1 was 22-fold higher in Snail-1 (*P<0.05) and 2.5-fold higher in SIP1-1 than control. Expression of MMP-2 was 8.7-fold higher in Snail-1 (*P<0.05) and 3.2-fold higher in SIP1-1. Expression of MMP-7 was 15.8-fold in Snail-1 (*P<0.05), MT1-MMP was 3.4-fold higher in Snail-1 (*P<0.05) and 2.7-fold higher in SIP1-1 (*P<0.05).
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References

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