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Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells.

Qian AR, Zhang W, Cao JP, Yang PF, Gao X, Wang Z, Xu HY, Weng YY, Shang P - J. Exp. Clin. Cancer Res. (2008)

Bottom Line: Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton.Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory for Space Biosciences & Biotechnology, Institute of Special Environmental Biophysics, Faculty of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, PR China. qianair@nwpu.edu.cn

ABSTRACT

Background: CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.

Methods: Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.

Results: Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression.

Conclusion: CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

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The effects of si-CD147 on SMMC-7721 microfilament actin, microtubule and vimentin. After being transfected with negative control or si-CD147 for 48 h, cells were trypsinized and cultured on coverslips, and then fixed. The effects of si-CD147 on SMMC-7721 cytoskeleton were investigated by confocal microscopy. Actin, mirotublin and vimentin filaments in si-CD147 transfected cells were significantly altered (A) and the mean signal intensities were quantified with ImageJ software (B). *P < 0.05 v.s. control.
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Figure 5: The effects of si-CD147 on SMMC-7721 microfilament actin, microtubule and vimentin. After being transfected with negative control or si-CD147 for 48 h, cells were trypsinized and cultured on coverslips, and then fixed. The effects of si-CD147 on SMMC-7721 cytoskeleton were investigated by confocal microscopy. Actin, mirotublin and vimentin filaments in si-CD147 transfected cells were significantly altered (A) and the mean signal intensities were quantified with ImageJ software (B). *P < 0.05 v.s. control.

Mentions: We examined the effects of siRNA-mediated CD147 gene silencing on SMMC-7721 cytoskeleton including actin filament, microtubule and intermediate filament by confocal microscopy. SMMC-7721 cells transfected with si-CD147 or control siRNA were labeled respectively with antibodies targeting actin, tublulin and vimentin to visualize these protein. In SMMC-7721 cells transfected with si-CD147, actin showed a nuclear concentration and decreased fluorescence intensity. (Figure. 5). The microtubule constructed to a network and cells appeared flattened in si-CD147 transfected cells, but the difference of fluorescence intensity between two groups was not significant (Figure. 5). Vimentin filaments showed an interlaced pattern in control cells but in transfected cells they seemed to form foci in the periphery of cells, and the difference of fluorescence intensity between two groups was not significant (Figure. 5).


Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells.

Qian AR, Zhang W, Cao JP, Yang PF, Gao X, Wang Z, Xu HY, Weng YY, Shang P - J. Exp. Clin. Cancer Res. (2008)

The effects of si-CD147 on SMMC-7721 microfilament actin, microtubule and vimentin. After being transfected with negative control or si-CD147 for 48 h, cells were trypsinized and cultured on coverslips, and then fixed. The effects of si-CD147 on SMMC-7721 cytoskeleton were investigated by confocal microscopy. Actin, mirotublin and vimentin filaments in si-CD147 transfected cells were significantly altered (A) and the mean signal intensities were quantified with ImageJ software (B). *P < 0.05 v.s. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572039&req=5

Figure 5: The effects of si-CD147 on SMMC-7721 microfilament actin, microtubule and vimentin. After being transfected with negative control or si-CD147 for 48 h, cells were trypsinized and cultured on coverslips, and then fixed. The effects of si-CD147 on SMMC-7721 cytoskeleton were investigated by confocal microscopy. Actin, mirotublin and vimentin filaments in si-CD147 transfected cells were significantly altered (A) and the mean signal intensities were quantified with ImageJ software (B). *P < 0.05 v.s. control.
Mentions: We examined the effects of siRNA-mediated CD147 gene silencing on SMMC-7721 cytoskeleton including actin filament, microtubule and intermediate filament by confocal microscopy. SMMC-7721 cells transfected with si-CD147 or control siRNA were labeled respectively with antibodies targeting actin, tublulin and vimentin to visualize these protein. In SMMC-7721 cells transfected with si-CD147, actin showed a nuclear concentration and decreased fluorescence intensity. (Figure. 5). The microtubule constructed to a network and cells appeared flattened in si-CD147 transfected cells, but the difference of fluorescence intensity between two groups was not significant (Figure. 5). Vimentin filaments showed an interlaced pattern in control cells but in transfected cells they seemed to form foci in the periphery of cells, and the difference of fluorescence intensity between two groups was not significant (Figure. 5).

Bottom Line: Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton.Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory for Space Biosciences & Biotechnology, Institute of Special Environmental Biophysics, Faculty of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, PR China. qianair@nwpu.edu.cn

ABSTRACT

Background: CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.

Methods: Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.

Results: Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression.

Conclusion: CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

Show MeSH
Related in: MedlinePlus