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A role for aberrantly expressed nuclear localized decorin in migration and invasion of dysplastic and malignant oral epithelial cells.

Dil N, Banerjee AG - Head Neck Oncol (2011)

Bottom Line: This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively.Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines.Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Oral Biology, University of Manitoba, Health Sciences Center, Winnipeg, Canada. dil@cc.umanitoba.ca

ABSTRACT

Background: Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression.

Materials and methods: We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines.

Results: More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia.

Conclusions: Taken together, our results indicate that nuclear localized decorin plays an important role in migration and invasion of oral cancer cells and thus may present as a novel potential target for the treatment of oral cancer.

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Migration and invasion suppression in decorin silenced cell lines. A, Cell motility through uncoated filters (migration) was measured 22 h after plating. The migrating cells were fixed, stained, and photographed as described in materials and methods. Each panel represents one representative field of five from duplicate filters of three experiments. B, Migrated cells in each one of the five fields of duplicate filters were counted, numbers represent mean ± SD of three experiments. C, Cells that invaded across the Matrigel™ layer were fixed, stained, and photographed. Each panel represents one representative field of five from duplicate filters of three experiments. D, Migrated and invaded cells in five fields of duplicate filters were counted and % invasion was calculated as described in materials and methods. Numbers represent mean ± SD of three individual experiments. ** p < 0.01, *** p < 0.001 compared to respective controls.
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Figure 3: Migration and invasion suppression in decorin silenced cell lines. A, Cell motility through uncoated filters (migration) was measured 22 h after plating. The migrating cells were fixed, stained, and photographed as described in materials and methods. Each panel represents one representative field of five from duplicate filters of three experiments. B, Migrated cells in each one of the five fields of duplicate filters were counted, numbers represent mean ± SD of three experiments. C, Cells that invaded across the Matrigel™ layer were fixed, stained, and photographed. Each panel represents one representative field of five from duplicate filters of three experiments. D, Migrated and invaded cells in five fields of duplicate filters were counted and % invasion was calculated as described in materials and methods. Numbers represent mean ± SD of three individual experiments. ** p < 0.01, *** p < 0.001 compared to respective controls.

Mentions: We examined whether decorin silencing has any effect on migration and invasion properties of dysplastic and malignant oral epithelial cells. Using an in vitro trans well assay and 10% FBS as a chemo-attractant, we observed a significant suppression of cell migration in both decorin-silenced DOK and SCC-25 cells compared to respective WT or control cells (Figure 3A &3B). Next, we determined the invasive property of these cells as measured through invasion across a Matrigel™ impregnated porous (8 μm) membrane. Invasive phenotype was observed to be significantly suppressed in decorin-silenced SCC-25 cells and was almost completely abrogated in decorin-silenced DOK cells (Figure 3C &3D). Similar results were obtained when conditioned media from DOK WT was used as a chemo-attractant (data not shown). It is important to note that overall malignant SCC-25 cells have relatively higher migration and invasion rates than the premalignant and dysplastic DOK cells.


A role for aberrantly expressed nuclear localized decorin in migration and invasion of dysplastic and malignant oral epithelial cells.

Dil N, Banerjee AG - Head Neck Oncol (2011)

Migration and invasion suppression in decorin silenced cell lines. A, Cell motility through uncoated filters (migration) was measured 22 h after plating. The migrating cells were fixed, stained, and photographed as described in materials and methods. Each panel represents one representative field of five from duplicate filters of three experiments. B, Migrated cells in each one of the five fields of duplicate filters were counted, numbers represent mean ± SD of three experiments. C, Cells that invaded across the Matrigel™ layer were fixed, stained, and photographed. Each panel represents one representative field of five from duplicate filters of three experiments. D, Migrated and invaded cells in five fields of duplicate filters were counted and % invasion was calculated as described in materials and methods. Numbers represent mean ± SD of three individual experiments. ** p < 0.01, *** p < 0.001 compared to respective controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198745&req=5

Figure 3: Migration and invasion suppression in decorin silenced cell lines. A, Cell motility through uncoated filters (migration) was measured 22 h after plating. The migrating cells were fixed, stained, and photographed as described in materials and methods. Each panel represents one representative field of five from duplicate filters of three experiments. B, Migrated cells in each one of the five fields of duplicate filters were counted, numbers represent mean ± SD of three experiments. C, Cells that invaded across the Matrigel™ layer were fixed, stained, and photographed. Each panel represents one representative field of five from duplicate filters of three experiments. D, Migrated and invaded cells in five fields of duplicate filters were counted and % invasion was calculated as described in materials and methods. Numbers represent mean ± SD of three individual experiments. ** p < 0.01, *** p < 0.001 compared to respective controls.
Mentions: We examined whether decorin silencing has any effect on migration and invasion properties of dysplastic and malignant oral epithelial cells. Using an in vitro trans well assay and 10% FBS as a chemo-attractant, we observed a significant suppression of cell migration in both decorin-silenced DOK and SCC-25 cells compared to respective WT or control cells (Figure 3A &3B). Next, we determined the invasive property of these cells as measured through invasion across a Matrigel™ impregnated porous (8 μm) membrane. Invasive phenotype was observed to be significantly suppressed in decorin-silenced SCC-25 cells and was almost completely abrogated in decorin-silenced DOK cells (Figure 3C &3D). Similar results were obtained when conditioned media from DOK WT was used as a chemo-attractant (data not shown). It is important to note that overall malignant SCC-25 cells have relatively higher migration and invasion rates than the premalignant and dysplastic DOK cells.

Bottom Line: This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively.Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines.Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Oral Biology, University of Manitoba, Health Sciences Center, Winnipeg, Canada. dil@cc.umanitoba.ca

ABSTRACT

Background: Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression.

Materials and methods: We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines.

Results: More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia.

Conclusions: Taken together, our results indicate that nuclear localized decorin plays an important role in migration and invasion of oral cancer cells and thus may present as a novel potential target for the treatment of oral cancer.

Show MeSH
Related in: MedlinePlus