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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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Detection of the effects of si-CD147 on gelatinase production by gelatin zymography (A, B) and western blot assay (C, D, E). The production and expression of gelatinase were detected by gelatin zymography (A, B) and western blot assay (C, D, E), after SMMC-7721 cells were treated with si-CD147 or negative control siRNA for 48 h. MMP-2 production was decreased significantly in SMMC-7721 cells transfected with si-CD147 (A). The graph (B) compares scanning signal intensity of MMP-2 by ImageJ software. MMP-2 and MMP-9 expression greatly decreased in SMMC-7721 cells transfected with si-CD147 (C). The graph (D, E) compares scanning signal intensity of MMP-9 and MMP-2 expression by ImageJ software.
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Figure 2: Detection of the effects of si-CD147 on gelatinase production by gelatin zymography (A, B) and western blot assay (C, D, E). The production and expression of gelatinase were detected by gelatin zymography (A, B) and western blot assay (C, D, E), after SMMC-7721 cells were treated with si-CD147 or negative control siRNA for 48 h. MMP-2 production was decreased significantly in SMMC-7721 cells transfected with si-CD147 (A). The graph (B) compares scanning signal intensity of MMP-2 by ImageJ software. MMP-2 and MMP-9 expression greatly decreased in SMMC-7721 cells transfected with si-CD147 (C). The graph (D, E) compares scanning signal intensity of MMP-9 and MMP-2 expression by ImageJ software.

Mentions: To investigate whether the downregulation of CD147 in SMMC-7721 affects MMPs secretion, we evaluated MMP-2 and MMP-9 expression in SMMC-7721 cells transfected with si-CD147 by gelatin zymography and western blot assay. Gelatin zymograpy analysis showed that MMP-2 production in SMMC-7721 cells transfected with si-CD147 was decreased by over 50% compared with that in negative control cells (Figure. 2A and 2B). Western blot analysis showed that both of the MMP-2 and MMP-9 expression were inhibited in SMMC-7721 cells transfected with si-CD147 compared with that in control cells (Figure. 2C, 2D and 2E).


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)

Detection of the effects of si-CD147 on gelatinase production by gelatin zymography (A, B) and western blot assay (C, D, E). The production and expression of gelatinase were detected by gelatin zymography (A, B) and western blot assay (C, D, E), after SMMC-7721 cells were treated with si-CD147 or negative control siRNA for 48 h. MMP-2 production was decreased significantly in SMMC-7721 cells transfected with si-CD147 (A). The graph (B) compares scanning signal intensity of MMP-2 by ImageJ software. MMP-2 and MMP-9 expression greatly decreased in SMMC-7721 cells transfected with si-CD147 (C). The graph (D, E) compares scanning signal intensity of MMP-9 and MMP-2 expression by ImageJ software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Detection of the effects of si-CD147 on gelatinase production by gelatin zymography (A, B) and western blot assay (C, D, E). The production and expression of gelatinase were detected by gelatin zymography (A, B) and western blot assay (C, D, E), after SMMC-7721 cells were treated with si-CD147 or negative control siRNA for 48 h. MMP-2 production was decreased significantly in SMMC-7721 cells transfected with si-CD147 (A). The graph (B) compares scanning signal intensity of MMP-2 by ImageJ software. MMP-2 and MMP-9 expression greatly decreased in SMMC-7721 cells transfected with si-CD147 (C). The graph (D, E) compares scanning signal intensity of MMP-9 and MMP-2 expression by ImageJ software.
Mentions: To investigate whether the downregulation of CD147 in SMMC-7721 affects MMPs secretion, we evaluated MMP-2 and MMP-9 expression in SMMC-7721 cells transfected with si-CD147 by gelatin zymography and western blot assay. Gelatin zymograpy analysis showed that MMP-2 production in SMMC-7721 cells transfected with si-CD147 was decreased by over 50% compared with that in negative control cells (Figure. 2A and 2B). Western blot analysis showed that both of the MMP-2 and MMP-9 expression were inhibited in SMMC-7721 cells transfected with si-CD147 compared with that in control cells (Figure. 2C, 2D and 2E).

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