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PMC42, a breast progenitor cancer cell line, has normal-like mRNA and microRNA transcriptomes.

Git A, Spiteri I, Blenkiron C, Dunning MJ, Pole JC, Chin SF, Wang Y, Smith J, Livesey FJ, Caldas C - Breast Cancer Res. (2008)

Bottom Line: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire.We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells.Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Breast Cancer Functional Genomics Laboratory, Cancer Research UK Cambridge Research Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. Anna.Git@cancer.org.uk

ABSTRACT

Introduction: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation.

Methods: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays, and real-time reverse transcription-polymerase chain reaction.

Results: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in-culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional, and post-transcriptional components.

Conclusion: We propose that, despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs that are typically lost in cancer.

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Validation of microRNA (miRNA) expression by Luminex bead arrays and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The levels of 26 miRNAs measured using the methods indicated on the left are shown. qRT-PCR experiments were performed three times, each in triplicate, except for miRNAs 7, 126, and 126* (red diamond), which were measured once in triplicate; error bars indicate one standard deviation. On Luminex bead arrays, miRNAs 127 and 17-3p were below detection (red star). As units of expression in all methods are arbitrary, data were scaled to the highest-expressing sample for each miRNA. Normalised slide microarray data are presented on a non-logarithmic scale identical to the other two methods, for which the data were not normalised in any way. PN miRNAs appear in boldface. miRNAs with at least one negative value for correlation between the methods are shown in red. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.
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Figure 3: Validation of microRNA (miRNA) expression by Luminex bead arrays and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The levels of 26 miRNAs measured using the methods indicated on the left are shown. qRT-PCR experiments were performed three times, each in triplicate, except for miRNAs 7, 126, and 126* (red diamond), which were measured once in triplicate; error bars indicate one standard deviation. On Luminex bead arrays, miRNAs 127 and 17-3p were below detection (red star). As units of expression in all methods are arbitrary, data were scaled to the highest-expressing sample for each miRNA. Normalised slide microarray data are presented on a non-logarithmic scale identical to the other two methods, for which the data were not normalised in any way. PN miRNAs appear in boldface. miRNAs with at least one negative value for correlation between the methods are shown in red. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.

Mentions: We validated the differential expression of the PN miRNAs by two independent methods for miRNA quantitation: single-channel Luminex bead arrays and real-time RT-PCR. Figure 3 summarises the results obtained from all three methods for 26 miRNAs, including 11 PN miRNAs as well as 15 non-PN control miRNAs with varying expression profiles. The alternative methods confirmed the specific loss of PN miRNAs, but not control miRNAs, from the non-PMC42 samples. The overall correlations between the methods were qRT-PCR/bead arrays 62%, qRT-PCR/slide arrays 65%, and bead arrays/slide arrays 43%. miRNAs 22 and 182 had at least one negative pair-wise correlation between the methods, and miR-126 appears to be expressed only in normal tissue, and these miRNAs were subsequently not considered as part of the PN cluster.


PMC42, a breast progenitor cancer cell line, has normal-like mRNA and microRNA transcriptomes.

Git A, Spiteri I, Blenkiron C, Dunning MJ, Pole JC, Chin SF, Wang Y, Smith J, Livesey FJ, Caldas C - Breast Cancer Res. (2008)

Validation of microRNA (miRNA) expression by Luminex bead arrays and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The levels of 26 miRNAs measured using the methods indicated on the left are shown. qRT-PCR experiments were performed three times, each in triplicate, except for miRNAs 7, 126, and 126* (red diamond), which were measured once in triplicate; error bars indicate one standard deviation. On Luminex bead arrays, miRNAs 127 and 17-3p were below detection (red star). As units of expression in all methods are arbitrary, data were scaled to the highest-expressing sample for each miRNA. Normalised slide microarray data are presented on a non-logarithmic scale identical to the other two methods, for which the data were not normalised in any way. PN miRNAs appear in boldface. miRNAs with at least one negative value for correlation between the methods are shown in red. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481505&req=5

Figure 3: Validation of microRNA (miRNA) expression by Luminex bead arrays and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The levels of 26 miRNAs measured using the methods indicated on the left are shown. qRT-PCR experiments were performed three times, each in triplicate, except for miRNAs 7, 126, and 126* (red diamond), which were measured once in triplicate; error bars indicate one standard deviation. On Luminex bead arrays, miRNAs 127 and 17-3p were below detection (red star). As units of expression in all methods are arbitrary, data were scaled to the highest-expressing sample for each miRNA. Normalised slide microarray data are presented on a non-logarithmic scale identical to the other two methods, for which the data were not normalised in any way. PN miRNAs appear in boldface. miRNAs with at least one negative value for correlation between the methods are shown in red. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.
Mentions: We validated the differential expression of the PN miRNAs by two independent methods for miRNA quantitation: single-channel Luminex bead arrays and real-time RT-PCR. Figure 3 summarises the results obtained from all three methods for 26 miRNAs, including 11 PN miRNAs as well as 15 non-PN control miRNAs with varying expression profiles. The alternative methods confirmed the specific loss of PN miRNAs, but not control miRNAs, from the non-PMC42 samples. The overall correlations between the methods were qRT-PCR/bead arrays 62%, qRT-PCR/slide arrays 65%, and bead arrays/slide arrays 43%. miRNAs 22 and 182 had at least one negative pair-wise correlation between the methods, and miR-126 appears to be expressed only in normal tissue, and these miRNAs were subsequently not considered as part of the PN cluster.

Bottom Line: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire.We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells.Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Breast Cancer Functional Genomics Laboratory, Cancer Research UK Cambridge Research Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. Anna.Git@cancer.org.uk

ABSTRACT

Introduction: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation.

Methods: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays, and real-time reverse transcription-polymerase chain reaction.

Results: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in-culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional, and post-transcriptional components.

Conclusion: We propose that, despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs that are typically lost in cancer.

Show MeSH
Related in: MedlinePlus