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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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Reduced IL-8 production in decorin Silenced DOK and SCC25. A, RNA was extracted from WT, control and decorin silenced DOK and SCC-25 cells and cDNA was subjected to quantitative RT-PCR, normalized IL-8 expression from one representative experiment of three. B, Cells were cultured and IL-8 was measured in 24 h culture supernatants using ELISA. Data are presented as mean ± SD of three replicates of one representative experiment of four. ** p < 0.01, *** p < 0.001 compared to respective controls.
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Figure 4: Reduced IL-8 production in decorin Silenced DOK and SCC25. A, RNA was extracted from WT, control and decorin silenced DOK and SCC-25 cells and cDNA was subjected to quantitative RT-PCR, normalized IL-8 expression from one representative experiment of three. B, Cells were cultured and IL-8 was measured in 24 h culture supernatants using ELISA. Data are presented as mean ± SD of three replicates of one representative experiment of four. ** p < 0.01, *** p < 0.001 compared to respective controls.

Mentions: IL-8 is an important pro-inflammatory chemokine and is involved in tumor progression in a variety of malignancies. In particular, IL-8 has been shown to induce migration and invasion in oral squamous cell carcinoma cell lines[23]. Moreover, decorin has been shown to induce IL-8 production in endothelial cells [25]. Therefore, we sought to determine if nuclear decorin silencing has an effect on IL-8 production in these dysplastic and malignant oral epithelial cells. Interestingly, IL-8 expression was significantly reduced in nuclear decorin-silenced DOK or SCC-25 cells as compared to the control and WT cells. Real-time PCR analysis revealed over 90% reduction in constitutive IL-8 expression in decorin-silenced DOK and about 70% reduction in decorin-silenced SCC-25 cells (Figure 4A). IL-8 protein production, as measured by ELISA, was found to be significantly reduced in decorin-silenced DOK and SCC-25 cells (Figure 4B). However, as observed with IL-8 expression levels, the effect of decorin silencing on IL-8 production was more pronounced in DOK than in SCC-25 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)

Reduced IL-8 production in decorin Silenced DOK and SCC25. A, RNA was extracted from WT, control and decorin silenced DOK and SCC-25 cells and cDNA was subjected to quantitative RT-PCR, normalized IL-8 expression from one representative experiment of three. B, Cells were cultured and IL-8 was measured in 24 h culture supernatants using ELISA. Data are presented as mean ± SD of three replicates of one representative experiment of four. ** 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 4: Reduced IL-8 production in decorin Silenced DOK and SCC25. A, RNA was extracted from WT, control and decorin silenced DOK and SCC-25 cells and cDNA was subjected to quantitative RT-PCR, normalized IL-8 expression from one representative experiment of three. B, Cells were cultured and IL-8 was measured in 24 h culture supernatants using ELISA. Data are presented as mean ± SD of three replicates of one representative experiment of four. ** p < 0.01, *** p < 0.001 compared to respective controls.
Mentions: IL-8 is an important pro-inflammatory chemokine and is involved in tumor progression in a variety of malignancies. In particular, IL-8 has been shown to induce migration and invasion in oral squamous cell carcinoma cell lines[23]. Moreover, decorin has been shown to induce IL-8 production in endothelial cells [25]. Therefore, we sought to determine if nuclear decorin silencing has an effect on IL-8 production in these dysplastic and malignant oral epithelial cells. Interestingly, IL-8 expression was significantly reduced in nuclear decorin-silenced DOK or SCC-25 cells as compared to the control and WT cells. Real-time PCR analysis revealed over 90% reduction in constitutive IL-8 expression in decorin-silenced DOK and about 70% reduction in decorin-silenced SCC-25 cells (Figure 4A). IL-8 protein production, as measured by ELISA, was found to be significantly reduced in decorin-silenced DOK and SCC-25 cells (Figure 4B). However, as observed with IL-8 expression levels, the effect of decorin silencing on IL-8 production was more pronounced in DOK than in SCC-25 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