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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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Validation of stable knockdown of decorin in DOK and SCC-25 cells. DOK and SCC-25 cells were stably transfected with decorin-shRNA (DCN-shRNA), or scrambled sequence-shRNA (Ctrl-shRNA) or no transfection control (WT). A, RNA was extracted and cDNA was subjected to quantitative RT-PCR, normalized decorin expression from one representative experiment of three. B, Nuclear lysates were extracted and were subjected to SDS-PAGE followed by immunoblotting with anti-decorin and anti-β-tubulin antibodies. Data presented is one representative immuoblot of at least three experiments. *** p < 0.001 compared to respective controls.
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Figure 1: Validation of stable knockdown of decorin in DOK and SCC-25 cells. DOK and SCC-25 cells were stably transfected with decorin-shRNA (DCN-shRNA), or scrambled sequence-shRNA (Ctrl-shRNA) or no transfection control (WT). A, RNA was extracted and cDNA was subjected to quantitative RT-PCR, normalized decorin expression from one representative experiment of three. B, Nuclear lysates were extracted and were subjected to SDS-PAGE followed by immunoblotting with anti-decorin and anti-β-tubulin antibodies. Data presented is one representative immuoblot of at least three experiments. *** p < 0.001 compared to respective controls.

Mentions: To study the functional role of aberrantly expressed nuclear decorin in dysplastic and malignant epithelial cells, decorin shRNA-stable clones were generated. Briefly, DNA oligonucleotides specific for decorin mRNA target sequence and a non-gene scrambled control were ligated into pGeneClip™ Puro plasmid, and are herein referred to as decorin shRNA (DCN-shRNA) and control shRNA (Ctrl-shRNA), respectively. DOK and SCC-25 cells were transfected with these constructs and puromycin resistant positive stable clones were selected. To avoid clone-specific effects, pooled transfectants were used for each cell type. Knockdown of decorin expression was confirmed by real-time PCR and western blot analysis. Pooled decorin-shRNA transfected DOK clones showed a significant (more than 80%) decrease in decorin mRNA expression when compared to control-shRNA transfected clones or no transfection wild type DOK (Figure 1A). Similar results were observed in SCC-25 cells (Figure 1A). Decorin knock down was also confirmed by western blot. Pooled decorin-shRNA transfected DOK or SCC-25 clones showed almost complete abrogation of decorin protein expression in nuclear lysates (Figure 1B). Similar decorin protein expression knock down was observed in whole cell lysates (data not shown). These results demonstrate that decorin-shRNA successfully silenced the nuclear decorin expression in DOK and 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)

Validation of stable knockdown of decorin in DOK and SCC-25 cells. DOK and SCC-25 cells were stably transfected with decorin-shRNA (DCN-shRNA), or scrambled sequence-shRNA (Ctrl-shRNA) or no transfection control (WT). A, RNA was extracted and cDNA was subjected to quantitative RT-PCR, normalized decorin expression from one representative experiment of three. B, Nuclear lysates were extracted and were subjected to SDS-PAGE followed by immunoblotting with anti-decorin and anti-β-tubulin antibodies. Data presented is one representative immuoblot of at least three experiments. *** 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 1: Validation of stable knockdown of decorin in DOK and SCC-25 cells. DOK and SCC-25 cells were stably transfected with decorin-shRNA (DCN-shRNA), or scrambled sequence-shRNA (Ctrl-shRNA) or no transfection control (WT). A, RNA was extracted and cDNA was subjected to quantitative RT-PCR, normalized decorin expression from one representative experiment of three. B, Nuclear lysates were extracted and were subjected to SDS-PAGE followed by immunoblotting with anti-decorin and anti-β-tubulin antibodies. Data presented is one representative immuoblot of at least three experiments. *** p < 0.001 compared to respective controls.
Mentions: To study the functional role of aberrantly expressed nuclear decorin in dysplastic and malignant epithelial cells, decorin shRNA-stable clones were generated. Briefly, DNA oligonucleotides specific for decorin mRNA target sequence and a non-gene scrambled control were ligated into pGeneClip™ Puro plasmid, and are herein referred to as decorin shRNA (DCN-shRNA) and control shRNA (Ctrl-shRNA), respectively. DOK and SCC-25 cells were transfected with these constructs and puromycin resistant positive stable clones were selected. To avoid clone-specific effects, pooled transfectants were used for each cell type. Knockdown of decorin expression was confirmed by real-time PCR and western blot analysis. Pooled decorin-shRNA transfected DOK clones showed a significant (more than 80%) decrease in decorin mRNA expression when compared to control-shRNA transfected clones or no transfection wild type DOK (Figure 1A). Similar results were observed in SCC-25 cells (Figure 1A). Decorin knock down was also confirmed by western blot. Pooled decorin-shRNA transfected DOK or SCC-25 clones showed almost complete abrogation of decorin protein expression in nuclear lysates (Figure 1B). Similar decorin protein expression knock down was observed in whole cell lysates (data not shown). These results demonstrate that decorin-shRNA successfully silenced the nuclear decorin expression in DOK and 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