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siRNA knockdown of ribosomal protein gene RPL19 abrogates the aggressive phenotype of human prostate cancer.

Bee A, Brewer D, Beesley C, Dodson A, Forootan S, Dickinson T, Gerard P, Lane B, Yao S, Cooper CS, Djamgoz MB, Gosden CM, Ke Y, Foster CS - PLoS ONE (2011)

Bottom Line: However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced.The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype.Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected.

View Article: PubMed Central - PubMed

Affiliation: Section of Cellular Pathology and Molecular Genetics, Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
We provide novel functional data that posttranscriptional silencing of gene RPL19 using RNAi not only abrogates the malignant phenotype of PC-3M prostate cancer cells but is selective with respect to transcription and translation of other genes. Reducing RPL19 transcription modulates a subset of genes, evidenced by gene expression array analysis and Western blotting, but does not compromise cell proliferation or apoptosis in-vitro. However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced. Analysis of the modulated genes reveals induction of the non-malignant phenotype principally to involve perturbation of networks of transcription factors and cellular adhesion genes. The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype. Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected.

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Graphical representation of gene expression modulated following RPL19 knockdown.Heat map of top 50 genes up-regulated and top50 genes down-regulated following expression-profiling of mRNA expressed by si-RPL19-PC-3Mclone ST-3 cells when compared to PC-3Mparental cells using PC-3Mscramble cells as the common denominator. Hierarchical clustering is shown. Green indicates genes over-expressed in a sample compared to scramble-transfected cells. Red indicates genes down-regulated in the sample when compared to scramble-transfected cells. Corresponding numerical data are presented in Supporting Information Tables S1 & S2.
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pone-0022672-g003: Graphical representation of gene expression modulated following RPL19 knockdown.Heat map of top 50 genes up-regulated and top50 genes down-regulated following expression-profiling of mRNA expressed by si-RPL19-PC-3Mclone ST-3 cells when compared to PC-3Mparental cells using PC-3Mscramble cells as the common denominator. Hierarchical clustering is shown. Green indicates genes over-expressed in a sample compared to scramble-transfected cells. Red indicates genes down-regulated in the sample when compared to scramble-transfected cells. Corresponding numerical data are presented in Supporting Information Tables S1 & S2.

Mentions: Genome-wide expression profiles obtained from DNA oligonucleotide microarrays (unmodified Agilent Human Genome 44K) were employed to identify genes modulated following RPL19 knockdown. Comparison of genes expressed by PC-3Mparental and PC-3Mscramble cell-lines revealed no statistically significant differences (p≥0.05), indicating that the transfection technique was not responsible for appreciable off-target effects that might bias the experimental data. A total of 916 DNA sequences, representing 768 genes, were identified as differentially expressed (p≤0.05, Benjamini and Hochberg multiple testing correction applied). Of these, 404 were enhanced and 364 down-regulated. Within that data set, 184 different genes were modulated at least four-fold, 62 being up-regulated and 122 down-regulated. The top 50 differentially-expressed genes in these two categories are summarized in Supporting Information Tables S1 and S2 and graphically (Figure 3). Expression data derived from the arrays were validated by qPCR providing independent quantifiable evidence of the magnitude and direction of change of individual genes. The observation that only 768 genes were modulated following RPL19 knockdown, with the levels of mRNA for a wide range of proteins either maintained or elevated, suggests that ribosomal protein RPL19 is differentially involved in protein synthesis rather than affecting all cellular protein synthesis in a non-specific manner.


siRNA knockdown of ribosomal protein gene RPL19 abrogates the aggressive phenotype of human prostate cancer.

Bee A, Brewer D, Beesley C, Dodson A, Forootan S, Dickinson T, Gerard P, Lane B, Yao S, Cooper CS, Djamgoz MB, Gosden CM, Ke Y, Foster CS - PLoS ONE (2011)

Graphical representation of gene expression modulated following RPL19 knockdown.Heat map of top 50 genes up-regulated and top50 genes down-regulated following expression-profiling of mRNA expressed by si-RPL19-PC-3Mclone ST-3 cells when compared to PC-3Mparental cells using PC-3Mscramble cells as the common denominator. Hierarchical clustering is shown. Green indicates genes over-expressed in a sample compared to scramble-transfected cells. Red indicates genes down-regulated in the sample when compared to scramble-transfected cells. Corresponding numerical data are presented in Supporting Information Tables S1 & S2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3142177&req=5

pone-0022672-g003: Graphical representation of gene expression modulated following RPL19 knockdown.Heat map of top 50 genes up-regulated and top50 genes down-regulated following expression-profiling of mRNA expressed by si-RPL19-PC-3Mclone ST-3 cells when compared to PC-3Mparental cells using PC-3Mscramble cells as the common denominator. Hierarchical clustering is shown. Green indicates genes over-expressed in a sample compared to scramble-transfected cells. Red indicates genes down-regulated in the sample when compared to scramble-transfected cells. Corresponding numerical data are presented in Supporting Information Tables S1 & S2.
Mentions: Genome-wide expression profiles obtained from DNA oligonucleotide microarrays (unmodified Agilent Human Genome 44K) were employed to identify genes modulated following RPL19 knockdown. Comparison of genes expressed by PC-3Mparental and PC-3Mscramble cell-lines revealed no statistically significant differences (p≥0.05), indicating that the transfection technique was not responsible for appreciable off-target effects that might bias the experimental data. A total of 916 DNA sequences, representing 768 genes, were identified as differentially expressed (p≤0.05, Benjamini and Hochberg multiple testing correction applied). Of these, 404 were enhanced and 364 down-regulated. Within that data set, 184 different genes were modulated at least four-fold, 62 being up-regulated and 122 down-regulated. The top 50 differentially-expressed genes in these two categories are summarized in Supporting Information Tables S1 and S2 and graphically (Figure 3). Expression data derived from the arrays were validated by qPCR providing independent quantifiable evidence of the magnitude and direction of change of individual genes. The observation that only 768 genes were modulated following RPL19 knockdown, with the levels of mRNA for a wide range of proteins either maintained or elevated, suggests that ribosomal protein RPL19 is differentially involved in protein synthesis rather than affecting all cellular protein synthesis in a non-specific manner.

Bottom Line: However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced.The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype.Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected.

View Article: PubMed Central - PubMed

Affiliation: Section of Cellular Pathology and Molecular Genetics, Department of Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.

ABSTRACT
We provide novel functional data that posttranscriptional silencing of gene RPL19 using RNAi not only abrogates the malignant phenotype of PC-3M prostate cancer cells but is selective with respect to transcription and translation of other genes. Reducing RPL19 transcription modulates a subset of genes, evidenced by gene expression array analysis and Western blotting, but does not compromise cell proliferation or apoptosis in-vitro. However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced. Analysis of the modulated genes reveals induction of the non-malignant phenotype principally to involve perturbation of networks of transcription factors and cellular adhesion genes. The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype. Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected.

Show MeSH
Related in: MedlinePlus