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Oncogenic Role of miR-15a-3p in 13q Amplicon-Driven Colorectal Adenoma-to-Carcinoma Progression.

de Groen FL, Timmer LM, Menezes RX, Diosdado B, Hooijberg E, Meijer GA, Steenbergen RD, Carvalho B - PLoS ONE (2015)

Bottom Line: A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development.These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells.Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands.

ABSTRACT
Progression from colorectal adenoma to carcinoma is strongly associated with an accumulation of genomic alterations, including gain of chromosome 13. This gain affects the whole q arm and is present in 40%-60% of all colorectal cancers (CRCs). Several genes located at this amplicon are known to be overexpressed in carcinomas due to copy number dosage. A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development. The present study set out to explore whether apart from mir-17~92, other miRNAs located at the 13q amplicon show a copy number dependent dosage effect that may contribute to 13q-driven colorectal adenoma-to-carcinoma progression. Integration of publically available miRNA expression, target mRNA expression and DNA copy number data from 125 CRCs yielded three miRNAs, miR-15a, -17, and -20a, of which high expression levels were significantly correlated with a 13q gain and which influenced target mRNA expression. These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells. Integration of miR-15a expression levels with mRNA expression data of predicted target genes identified mitochondrial uncoupling protein 2 (UCP2) and COP9 signalosome subunit 2 (COPS2) as candidates with significantly decreased expression in CRCs with 13q gain. Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression. In conclusion, significant overexpression of miR-15a-3p due to gain of 13q is functionally relevant in CRC, with UCP2 and COPS2 as candidate target genes. Taken together our findings suggest that miR-15a-3p may contribute to adenoma-to-carcinoma progression.

No MeSH data available.


Related in: MedlinePlus

Functional analysis of the effect of miR-15a-5p, -3p and miR-17 silencing in CRC cell line SW480.A) Representative example of expression levels of miRNAs upon silencing using antagomirs compared to a non-targeting control (**p<0.01; *p<0.05). B) Cell viability determined by MTT assay upon miRNA silencing in SW480 compared to non-targeting control (* p<0.05).
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pone.0132495.g003: Functional analysis of the effect of miR-15a-5p, -3p and miR-17 silencing in CRC cell line SW480.A) Representative example of expression levels of miRNAs upon silencing using antagomirs compared to a non-targeting control (**p<0.01; *p<0.05). B) Cell viability determined by MTT assay upon miRNA silencing in SW480 compared to non-targeting control (* p<0.05).

Mentions: The antagomirs were introduced into the SW480 CRC cell line, which has a gain of 13q encompassing the miR-15a locus. Efficiency of silencing varied per experiment between 75%-97%, 60–80% and 94%-96% for miR-15a-5p, miR-15a-3p and miR-17, respectively (Fig 3A). Silencing of miR-15a-3p and miR-17 led to a significant decrease in cell viability (Fig 3B). Silencing of miR-15a-5p also decreased cell viability significantly, although results were less consistent throughout replicate experiments (data not shown).


Oncogenic Role of miR-15a-3p in 13q Amplicon-Driven Colorectal Adenoma-to-Carcinoma Progression.

de Groen FL, Timmer LM, Menezes RX, Diosdado B, Hooijberg E, Meijer GA, Steenbergen RD, Carvalho B - PLoS ONE (2015)

Functional analysis of the effect of miR-15a-5p, -3p and miR-17 silencing in CRC cell line SW480.A) Representative example of expression levels of miRNAs upon silencing using antagomirs compared to a non-targeting control (**p<0.01; *p<0.05). B) Cell viability determined by MTT assay upon miRNA silencing in SW480 compared to non-targeting control (* p<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492490&req=5

pone.0132495.g003: Functional analysis of the effect of miR-15a-5p, -3p and miR-17 silencing in CRC cell line SW480.A) Representative example of expression levels of miRNAs upon silencing using antagomirs compared to a non-targeting control (**p<0.01; *p<0.05). B) Cell viability determined by MTT assay upon miRNA silencing in SW480 compared to non-targeting control (* p<0.05).
Mentions: The antagomirs were introduced into the SW480 CRC cell line, which has a gain of 13q encompassing the miR-15a locus. Efficiency of silencing varied per experiment between 75%-97%, 60–80% and 94%-96% for miR-15a-5p, miR-15a-3p and miR-17, respectively (Fig 3A). Silencing of miR-15a-3p and miR-17 led to a significant decrease in cell viability (Fig 3B). Silencing of miR-15a-5p also decreased cell viability significantly, although results were less consistent throughout replicate experiments (data not shown).

Bottom Line: A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development.These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells.Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands.

ABSTRACT
Progression from colorectal adenoma to carcinoma is strongly associated with an accumulation of genomic alterations, including gain of chromosome 13. This gain affects the whole q arm and is present in 40%-60% of all colorectal cancers (CRCs). Several genes located at this amplicon are known to be overexpressed in carcinomas due to copy number dosage. A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development. The present study set out to explore whether apart from mir-17~92, other miRNAs located at the 13q amplicon show a copy number dependent dosage effect that may contribute to 13q-driven colorectal adenoma-to-carcinoma progression. Integration of publically available miRNA expression, target mRNA expression and DNA copy number data from 125 CRCs yielded three miRNAs, miR-15a, -17, and -20a, of which high expression levels were significantly correlated with a 13q gain and which influenced target mRNA expression. These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells. Integration of miR-15a expression levels with mRNA expression data of predicted target genes identified mitochondrial uncoupling protein 2 (UCP2) and COP9 signalosome subunit 2 (COPS2) as candidates with significantly decreased expression in CRCs with 13q gain. Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression. In conclusion, significant overexpression of miR-15a-3p due to gain of 13q is functionally relevant in CRC, with UCP2 and COPS2 as candidate target genes. Taken together our findings suggest that miR-15a-3p may contribute to adenoma-to-carcinoma progression.

No MeSH data available.


Related in: MedlinePlus