The interaction of HAb18G/CD147 with integrin alpha6beta1 and its implications for the invasion potential of human hepatoma cells

Abstract

Background: HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin alpha3beta1. However, it has never been investigated whether alpha3beta1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells.

Methods: Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin alpha6beta1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin alpha6beta1. Invasion potential was evaluated with an invasion assay and gelatin zymography.

Results: We found that integrin alpha6beta1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin alpha6beta1 antibodies (P < 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P < 0.05). Importantly, no additive effect between Wortmannin and alpha6beta1 antibodies was observed, indicating that alpha6beta1 and PI3K transmit the signal in an upstream-downstream relationship.

Conclusion: These results suggest that alpha6beta1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

Figure 1

Expression and co-localization of HAb18G/CD147 with integrin α6 and β1 subunits in FHCC98 cells. FHCC98 cells were double-stained for HAb18G/CD147 (red) and integrin α6 or β1 (green).

Figure 2

Immunoprecipitation of HAb18G/CD147 and integrin α6 and β1 subunits in FHCC98 cells. (A) Precipitates from HAb18G/CD147 immunocomplexes were assayed for precipitated integrin α6 and β1 subunits. Precipitates from irrelevant antibody (Mouse IgG) were used as a negative control. (B) Expression of HAb18G/CD147 in FHCC98 cell lysates by Western blot. (C) Expression of integrin α6 and β1 subunits in FHCC98 cell lysates by Western blot. (D) Precipitates from α6β1 immunocomplexes were assayed for precipitated HAb18G/CD147. Precipitates from irrelevant antibody (Mouse IgG) were used as a negative control.

Figure 3

Effect of silencing HAb18G/CD147 in FHCC98 and 7721 cells. Forty-eight hours after siCD147 or sncRNA transfection of FHCC98 and 7721 cells, HAb18G/CD147 expression levels were examined by RT-PCR (A) and Western blot (B).

Figure 4

Invasion of hepatoma cells with or without integrin α6β1 mAbs and siHAb18G/CD147 treatment. (A-D) Matrigel invasion assay of FHCC98 (A, B) and 7721 (C, D) cells. Bars represent the mean of triplicate samples; error bars represent standard deviation. Data are representative of three independent experiments. ***P < 0.01 versus corresponding cells with no antibody treatment.

Figure 5

MMP secretion of hepatoma cells with or without integrin α6β1 mAbs and siHAb18G/CD147 treatment. MMP levels of FHCC98 (A, B) and 7721 (A, C) cells. Bars represent the mean of triplicate samples; error bars represent standard deviation. Data are representative of three independent experiments. ***P < 0.01, * P < 0.05 versus corresponding cells with no antibody treatment.

Figure 6

Effects of a PI3K inhibitor on invasion and MMP secretion of FHCC98 and 7721 cells. (A-C) Matrigel invasion assay of FHCC98 (A, B) and 7721 (A, C) cells. (D-F) MMP levels of FHCC98 (D, E) and 7721 (D, F) cells. Bars represent the mean of triplicate samples; error bars represent standard deviation. Data are representative of three independent experiments. * P < 0.05 versus corresponding untreated cells.

Figure 7

Akt phosphorylation in FHCC98 and 7721 cells. Forty-eight hours after siCD147 or sncRNA transfection of FHCC98 and 7721 cells, phospho-Akt and total-Akt expression levels were examined by Western blot. Bars represent the mean of triplicate samples; error bars represent standard deviation. Data are representative of three independent experiments. ** P < 0.01 versus corresponding controls.