Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer

Lise Lotte Christensen; Anja Holm; Juha Rantala; Olli Kallioniemi; Mads H Rasmussen; Marie S Ostenfeld; Frederik Dagnaes-Hansen; Bodil Oster; Troels Schepeler; Heidi Tobiasen; Kasper Thorsen; Oliver M Sieber; Peter Gibbs; Philippe Lamy; Torben F Hansen; Anders Jakobsen; Eva M Riising; Kristian Helin; Jan Lubinski; Rikke Hagemann-Madsen; Søren Laurberg; Torben F Orntoft; Claus L Andersen

doi:10.1371/journal.pone.0096767

Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer

Lise Lotte Christensen, Anja Holm, Juha Rantala, Olli Kallioniemi, Mads H Rasmussen, Marie S Ostenfeld, Frederik Dagnaes-Hansen, Bodil Oster, Troels Schepeler, Heidi Tobiasen, Kasper Thorsen, Oliver M Sieber, Peter Gibbs, Philippe Lamy, Torben F Hansen, Anders Jakobsen, Eva M Riising, Kristian Helin, Jan Lubinski, Rikke Hagemann-MadsenSøren Laurberg, Torben F Orntoft, Claus L Andersen

Department of Clinical Medicine

46 Citations (Scopus)

Abstract

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

Original language	English
Journal	PLOS ONE
Volume	9
Issue number	6
Pages (from-to)	e96767
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0096767
Publication status	Published - 3 Jun 2014

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Christensen, L. L., Holm, A., Rantala, J., Kallioniemi, O., Rasmussen, M. H., Ostenfeld, M. S., Dagnaes-Hansen, F., Oster, B., Schepeler, T., Tobiasen, H., Thorsen, K., Sieber, O. M., Gibbs, P., Lamy, P., Hansen, T. F., Jakobsen, A., Riising, E. M., Helin, K., Lubinski, J., ... Andersen, C. L. (2014). Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer. PLOS ONE, 9(6), e96767. https://doi.org/10.1371/journal.pone.0096767

Christensen, LL, Holm, A, Rantala, J, Kallioniemi, O, Rasmussen, MH, Ostenfeld, MS, Dagnaes-Hansen, F, Oster, B, Schepeler, T, Tobiasen, H, Thorsen, K, Sieber, OM, Gibbs, P, Lamy, P, Hansen, TF, Jakobsen, A, Riising, EM, Helin, K, Lubinski, J, Hagemann-Madsen, R, Laurberg, S, Orntoft, TF & Andersen, CL 2014, 'Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer', PLOS ONE, vol. 9, no. 6, pp. e96767. https://doi.org/10.1371/journal.pone.0096767

@article{60e846f7721d4d8cafc701bc624283a6,

title = "Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer",

abstract = "MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.",

author = "Christensen, {Lise Lotte} and Anja Holm and Juha Rantala and Olli Kallioniemi and Rasmussen, {Mads H} and Ostenfeld, {Marie S} and Frederik Dagnaes-Hansen and Bodil Oster and Troels Schepeler and Heidi Tobiasen and Kasper Thorsen and Sieber, {Oliver M} and Peter Gibbs and Philippe Lamy and Hansen, {Torben F} and Anders Jakobsen and Riising, {Eva M} and Kristian Helin and Jan Lubinski and Rikke Hagemann-Madsen and S{\o}ren Laurberg and Orntoft, {Torben F} and Andersen, {Claus L}",

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doi = "10.1371/journal.pone.0096767",

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T1 - Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer

AU - Christensen, Lise Lotte

AU - Holm, Anja

AU - Rantala, Juha

AU - Kallioniemi, Olli

AU - Rasmussen, Mads H

AU - Ostenfeld, Marie S

AU - Dagnaes-Hansen, Frederik

AU - Oster, Bodil

AU - Schepeler, Troels

AU - Tobiasen, Heidi

AU - Thorsen, Kasper

AU - Sieber, Oliver M

AU - Gibbs, Peter

AU - Lamy, Philippe

AU - Hansen, Torben F

AU - Jakobsen, Anders

AU - Riising, Eva M

AU - Helin, Kristian

AU - Lubinski, Jan

AU - Hagemann-Madsen, Rikke

AU - Laurberg, Søren

AU - Orntoft, Torben F

AU - Andersen, Claus L

PY - 2014/6/3

Y1 - 2014/6/3

N2 - MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

AB - MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

U2 - 10.1371/journal.pone.0096767

DO - 10.1371/journal.pone.0096767

M3 - Journal article

C2 - 24892549

SN - 1932-6203

VL - 9

SP - e96767

JO - PLoS Computational Biology

JF - PLoS Computational Biology

IS - 6

ER -

Functional Screening Identifies miRNAs Influencing Apoptosis and Proliferation in Colorectal Cancer

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