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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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Related in: MedlinePlus

Effects of si-CD147RNA on SMMC-7721 cells adhesion to fibronectin, collagen IV and laminin. SMMC-7721 cells (5 × 104) transfected with negative control or si-CD147 were added to 96-well micro-plates coated with FN, LN, and collagen IV. The cells were allowed to adhere for 60 min at 37°C and were then stained with 0.5% crystal violet in 20% (v/v) methanol/water. The amount of bound cells was estimated by solubilizing the dye using 0.1 M sodium citrate and reading the absorbance at 490 nm. The results indicated that si-CD147 inhibited SMMC-7721 adhesion to collagen IV but had no effects on adhesion to FN and LN. *P < 0.05 v.s. control.
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Figure 4: Effects of si-CD147RNA on SMMC-7721 cells adhesion to fibronectin, collagen IV and laminin. SMMC-7721 cells (5 × 104) transfected with negative control or si-CD147 were added to 96-well micro-plates coated with FN, LN, and collagen IV. The cells were allowed to adhere for 60 min at 37°C and were then stained with 0.5% crystal violet in 20% (v/v) methanol/water. The amount of bound cells was estimated by solubilizing the dye using 0.1 M sodium citrate and reading the absorbance at 490 nm. The results indicated that si-CD147 inhibited SMMC-7721 adhesion to collagen IV but had no effects on adhesion to FN and LN. *P < 0.05 v.s. control.

Mentions: The effects of si-CD147 on the adhesive abilities of HCC cells to ECM proteins including FN, LN and collagne IV were detected by adhesion assays. The results indicated that si-CD147 inhibited SMMC-7721 adhesion to collagen IV but had no effects on SMMC-7721 cells adhesion to FN and LN (Figure. 4).


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)

Effects of si-CD147RNA on SMMC-7721 cells adhesion to fibronectin, collagen IV and laminin. SMMC-7721 cells (5 × 104) transfected with negative control or si-CD147 were added to 96-well micro-plates coated with FN, LN, and collagen IV. The cells were allowed to adhere for 60 min at 37°C and were then stained with 0.5% crystal violet in 20% (v/v) methanol/water. The amount of bound cells was estimated by solubilizing the dye using 0.1 M sodium citrate and reading the absorbance at 490 nm. The results indicated that si-CD147 inhibited SMMC-7721 adhesion to collagen IV but had no effects on adhesion to FN and LN. *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 4: Effects of si-CD147RNA on SMMC-7721 cells adhesion to fibronectin, collagen IV and laminin. SMMC-7721 cells (5 × 104) transfected with negative control or si-CD147 were added to 96-well micro-plates coated with FN, LN, and collagen IV. The cells were allowed to adhere for 60 min at 37°C and were then stained with 0.5% crystal violet in 20% (v/v) methanol/water. The amount of bound cells was estimated by solubilizing the dye using 0.1 M sodium citrate and reading the absorbance at 490 nm. The results indicated that si-CD147 inhibited SMMC-7721 adhesion to collagen IV but had no effects on adhesion to FN and LN. *P < 0.05 v.s. control.
Mentions: The effects of si-CD147 on the adhesive abilities of HCC cells to ECM proteins including FN, LN and collagne IV were detected by adhesion assays. The results indicated that si-CD147 inhibited SMMC-7721 adhesion to collagen IV but had no effects on SMMC-7721 cells adhesion to FN and LN (Figure. 4).

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