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A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts.

Suh EJ, Remillard MY, Legesse-Miller A, Johnson EL, Lemons JM, Chapman TR, Forman JJ, Kojima M, Silberman ES, Coller HA - Genome Biol. (2012)

Bottom Line: In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence.We also found that let-7 and miR-125 were upregulated in quiescent cells.Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further. microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.

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ABSTRACT

Background: Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.

Results: Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.

Conclusions: microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.

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Widespread changes in microRNA abundance with quiescence. (A) The log2 fold-change in the expression of the 142 microRNAs that change expression at a 1% FDR during serum starvation (SS) or contact inhibition (CI) are depicted with respect to their average expression in proliferating (P) cells. Blue and yellow indicate negative and positive values, respectively. Genes are in order of the magnitude of their mean log2 fold change from proliferation to quiescence. (B) 'Volcano' plot of microRNA average log2 fold-change in quiescence conditions on the x-axis versus the log10 P value for the significance of the quiescence parameter in gene expression on the y-axis.
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Figure 1: Widespread changes in microRNA abundance with quiescence. (A) The log2 fold-change in the expression of the 142 microRNAs that change expression at a 1% FDR during serum starvation (SS) or contact inhibition (CI) are depicted with respect to their average expression in proliferating (P) cells. Blue and yellow indicate negative and positive values, respectively. Genes are in order of the magnitude of their mean log2 fold change from proliferation to quiescence. (B) 'Volcano' plot of microRNA average log2 fold-change in quiescence conditions on the x-axis versus the log10 P value for the significance of the quiescence parameter in gene expression on the y-axis.

Mentions: We have developed a model system of quiescence in primary human fibroblasts in which quiescence can be induced by either serum starvation or contact inhibition. Either condition results in an accumulation of quiescent cells, as indicated by cell cycle markers and RNA content [52]. Using one-color microRNA microarrays, we monitored microRNA expression levels in proliferating, serum-starved, and contact-inhibited primary human dermal fibroblasts (Figure 1A). Among the 209 microRNAs detected above background, 142 (68%) were expressed at different levels in proliferating compared with either serum-starved or contact-inhibited fibroblasts at a false-discovery rate of 1% (Figure 1B). microRNA expression patterns for contact inhibition and serum starvation were extremely similar, with a 95% confidence interval (CI) Pearson's correlation of 0.952 to 0.975, much more so than the mRNA expression patterns for the same conditions (Pearson's correlation of 0.319 to 0.341, 95% CI) (Additional File 1, Figure S1). This large difference in the amount of correlation between quiescence states may be due to experimental design or microarray platform differences, but an alternative explanation is that microRNAs exhibit more of a common quiescence signature than protein-coding transcripts [2]. microRNAs downregulated in quiescent cells included miR-18, miR-20, miR-29, and miR-7, and microRNAs upregulated with quiescence included let-7b, miR-125a, miR-30, miR-181, miR-26, and miR-199. With a stringent cutoff of greater than two-fold expression change due to quiescence, eight microRNAs were expressed at higher levels in proliferating cells and eight were expressed at higher levels in quiescent cells (Additional File 1, Table S1).


A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts.

Suh EJ, Remillard MY, Legesse-Miller A, Johnson EL, Lemons JM, Chapman TR, Forman JJ, Kojima M, Silberman ES, Coller HA - Genome Biol. (2012)

Widespread changes in microRNA abundance with quiescence. (A) The log2 fold-change in the expression of the 142 microRNAs that change expression at a 1% FDR during serum starvation (SS) or contact inhibition (CI) are depicted with respect to their average expression in proliferating (P) cells. Blue and yellow indicate negative and positive values, respectively. Genes are in order of the magnitude of their mean log2 fold change from proliferation to quiescence. (B) 'Volcano' plot of microRNA average log2 fold-change in quiescence conditions on the x-axis versus the log10 P value for the significance of the quiescence parameter in gene expression on the y-axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924601&req=5

Figure 1: Widespread changes in microRNA abundance with quiescence. (A) The log2 fold-change in the expression of the 142 microRNAs that change expression at a 1% FDR during serum starvation (SS) or contact inhibition (CI) are depicted with respect to their average expression in proliferating (P) cells. Blue and yellow indicate negative and positive values, respectively. Genes are in order of the magnitude of their mean log2 fold change from proliferation to quiescence. (B) 'Volcano' plot of microRNA average log2 fold-change in quiescence conditions on the x-axis versus the log10 P value for the significance of the quiescence parameter in gene expression on the y-axis.
Mentions: We have developed a model system of quiescence in primary human fibroblasts in which quiescence can be induced by either serum starvation or contact inhibition. Either condition results in an accumulation of quiescent cells, as indicated by cell cycle markers and RNA content [52]. Using one-color microRNA microarrays, we monitored microRNA expression levels in proliferating, serum-starved, and contact-inhibited primary human dermal fibroblasts (Figure 1A). Among the 209 microRNAs detected above background, 142 (68%) were expressed at different levels in proliferating compared with either serum-starved or contact-inhibited fibroblasts at a false-discovery rate of 1% (Figure 1B). microRNA expression patterns for contact inhibition and serum starvation were extremely similar, with a 95% confidence interval (CI) Pearson's correlation of 0.952 to 0.975, much more so than the mRNA expression patterns for the same conditions (Pearson's correlation of 0.319 to 0.341, 95% CI) (Additional File 1, Figure S1). This large difference in the amount of correlation between quiescence states may be due to experimental design or microarray platform differences, but an alternative explanation is that microRNAs exhibit more of a common quiescence signature than protein-coding transcripts [2]. microRNAs downregulated in quiescent cells included miR-18, miR-20, miR-29, and miR-7, and microRNAs upregulated with quiescence included let-7b, miR-125a, miR-30, miR-181, miR-26, and miR-199. With a stringent cutoff of greater than two-fold expression change due to quiescence, eight microRNAs were expressed at higher levels in proliferating cells and eight were expressed at higher levels in quiescent cells (Additional File 1, Table S1).

Bottom Line: In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence.We also found that let-7 and miR-125 were upregulated in quiescent cells.Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further. microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Although quiescence (reversible cell cycle arrest) is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.

Results: Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.

Conclusions: microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.

Show MeSH
Related in: MedlinePlus