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Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.

Teferedegne B, Murata H, Quiñones M, Peden K, Lewis AM - PLoS ONE (2010)

Bottom Line: When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells.These results may prove to be relevant to the biology of neoplastic development.In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of DNA Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT
MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

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Hierarchical clustering of miRNA expression.miRNAs included in the heat-map had a fold change >±2 and were significantly expressed (p<0.01). Each row shows the relative expression level for a single miRNA and each column represents miRNA profiles of the average triplicate array data: (1) pAGMK cells; (2) 10-87 LP cells; (3) 10-87 HP cells; (4) 10-87 T cells. The red or green color indicates relative high or low expression, respectively. Expression clusters representing different patterns of up-regulation to down-regulation are depicted by Roman numerals on the right hand side of the Figure.
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pone-0014416-g003: Hierarchical clustering of miRNA expression.miRNAs included in the heat-map had a fold change >±2 and were significantly expressed (p<0.01). Each row shows the relative expression level for a single miRNA and each column represents miRNA profiles of the average triplicate array data: (1) pAGMK cells; (2) 10-87 LP cells; (3) 10-87 HP cells; (4) 10-87 T cells. The red or green color indicates relative high or low expression, respectively. Expression clusters representing different patterns of up-regulation to down-regulation are depicted by Roman numerals on the right hand side of the Figure.

Mentions: The dysregulation of miRNA expression has been observed in cells obtained from various cancers [13], [20], [22]. Because the serial passage of VERO cells results in the acquisition of a tumorigenic phenotype [11], we evaluated whether miRNA expression could be correlated with the evolution of the neoplastic processes that were occurring in VERO cells during serial tissue-culture passage. Array-based miRNA profiling was performed on pAGMK cells and on non-tumorigenic and tumorigenic VERO cells. For each cell type, the analysis was performed in triplicate. Out of 776 primate miRNAs assayed, 654 were detected above background levels in at least 3 of the 12 datasets. When unsupervised hierarchical clustering analysis was done on the 654 miRNAs expressed in pAGMK cells, 10-87 LP cells, 10-87 HP cells, or 10-87 T cells, the miRNA tree generated was able to discriminate among all four types of cells, with pAGMK cells located at a distance from the clusters of the three VERO cell line derivatives (data not shown). ANOVA was done using the four cell types. All significantly expressed miRNAs with mean intensity values of more than 500 (arbitrary units) in at least in one of the four cell types and a p<0.01 were selected and transformed to z-intensity values for inclusion in the heat-maps. These miRNAs were regarded as significant and were considered to be differentially expressed (Fig. 3). Four distinct clusters of miRNAs were evident according to their similar expression patterns. The expression profile of miRNA in cluster-I indicated a general up-regulation in tumorigenic 10-87 HP cells and 10-87 T cells compared with pAGMK cells and 10-87 LP cells. Clusters-II,-III, and -IV contained miRNAs that were down-regulated in 10-87 HP cells and 10-87 T cells. Expression readouts of the miRNA arrays were subjected to paired, volcano-plot analysis to arrive at differences of means for each miRNA in 10-87 LP cells compared with 10-87 HP cells and 10-87 T cells, and 10-87 HP cells compared with 10-87 T cells. The analysis revealed distinct patterns of miRNA expression that distinguished the non-tumorigenic 10-87 LP cells from the tumorigenic 10-87 HP cells and 10-87 T cells (data not shown). When this comparison was performed between the miRNA expression profiles of 10-87 HP cells and 10-87 T cells, no significant differences were apparent (Table 2). These observations indicated that the dysregulation of miRNA expression during in vitro passaging correlated with the conversion of 10-87 LP cells to a tumorigenic phenotype at higher passage levels. Furthermore, no additional dysregulation of miRNA expression appeared to occur during tumor formation.


Patterns of microRNA expression in non-human primate cells correlate with neoplastic development in vitro.

Teferedegne B, Murata H, Quiñones M, Peden K, Lewis AM - PLoS ONE (2010)

Hierarchical clustering of miRNA expression.miRNAs included in the heat-map had a fold change >±2 and were significantly expressed (p<0.01). Each row shows the relative expression level for a single miRNA and each column represents miRNA profiles of the average triplicate array data: (1) pAGMK cells; (2) 10-87 LP cells; (3) 10-87 HP cells; (4) 10-87 T cells. The red or green color indicates relative high or low expression, respectively. Expression clusters representing different patterns of up-regulation to down-regulation are depicted by Roman numerals on the right hand side of the Figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008671&req=5

pone-0014416-g003: Hierarchical clustering of miRNA expression.miRNAs included in the heat-map had a fold change >±2 and were significantly expressed (p<0.01). Each row shows the relative expression level for a single miRNA and each column represents miRNA profiles of the average triplicate array data: (1) pAGMK cells; (2) 10-87 LP cells; (3) 10-87 HP cells; (4) 10-87 T cells. The red or green color indicates relative high or low expression, respectively. Expression clusters representing different patterns of up-regulation to down-regulation are depicted by Roman numerals on the right hand side of the Figure.
Mentions: The dysregulation of miRNA expression has been observed in cells obtained from various cancers [13], [20], [22]. Because the serial passage of VERO cells results in the acquisition of a tumorigenic phenotype [11], we evaluated whether miRNA expression could be correlated with the evolution of the neoplastic processes that were occurring in VERO cells during serial tissue-culture passage. Array-based miRNA profiling was performed on pAGMK cells and on non-tumorigenic and tumorigenic VERO cells. For each cell type, the analysis was performed in triplicate. Out of 776 primate miRNAs assayed, 654 were detected above background levels in at least 3 of the 12 datasets. When unsupervised hierarchical clustering analysis was done on the 654 miRNAs expressed in pAGMK cells, 10-87 LP cells, 10-87 HP cells, or 10-87 T cells, the miRNA tree generated was able to discriminate among all four types of cells, with pAGMK cells located at a distance from the clusters of the three VERO cell line derivatives (data not shown). ANOVA was done using the four cell types. All significantly expressed miRNAs with mean intensity values of more than 500 (arbitrary units) in at least in one of the four cell types and a p<0.01 were selected and transformed to z-intensity values for inclusion in the heat-maps. These miRNAs were regarded as significant and were considered to be differentially expressed (Fig. 3). Four distinct clusters of miRNAs were evident according to their similar expression patterns. The expression profile of miRNA in cluster-I indicated a general up-regulation in tumorigenic 10-87 HP cells and 10-87 T cells compared with pAGMK cells and 10-87 LP cells. Clusters-II,-III, and -IV contained miRNAs that were down-regulated in 10-87 HP cells and 10-87 T cells. Expression readouts of the miRNA arrays were subjected to paired, volcano-plot analysis to arrive at differences of means for each miRNA in 10-87 LP cells compared with 10-87 HP cells and 10-87 T cells, and 10-87 HP cells compared with 10-87 T cells. The analysis revealed distinct patterns of miRNA expression that distinguished the non-tumorigenic 10-87 LP cells from the tumorigenic 10-87 HP cells and 10-87 T cells (data not shown). When this comparison was performed between the miRNA expression profiles of 10-87 HP cells and 10-87 T cells, no significant differences were apparent (Table 2). These observations indicated that the dysregulation of miRNA expression during in vitro passaging correlated with the conversion of 10-87 LP cells to a tumorigenic phenotype at higher passage levels. Furthermore, no additional dysregulation of miRNA expression appeared to occur during tumor formation.

Bottom Line: When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells.These results may prove to be relevant to the biology of neoplastic development.In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of DNA Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.

ABSTRACT
MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype.

Show MeSH
Related in: MedlinePlus