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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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Changes in target genes with quiescence. (A) Hierarchical clustered heat map representing the log2 fold change of gene expression for all 15,560 consistently detectable genes during 1, 2, 4, 8, 24, and 96 h of serum starvation (SS), 1, 2, 4, 8, 24, and 48 h serum restimulation (SR), and 7 and 14 days (each repeated twice) of contact inhibition (CI). Expression in serum starvation and contact inhibition is shown relative to proliferating cells, and expression during serum restimulation is shown relative to 4-day serum-starved cells. Colors are as in Figure 1A. Numerals designate 4 different clusters chosen from the hierarchical clustering tree. Select enriched gene ontology terms for each of the clusters are shown in Additional File 1, Table S2. (B) Volcano plot of the mean projection of the microRNA target genes' log2 expression onto the array's first eigengene (Additional File 1, Figure S3B) on the x-axis versus the log10 P value of the mean projection on the y-axis.
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Figure 2: Changes in target genes with quiescence. (A) Hierarchical clustered heat map representing the log2 fold change of gene expression for all 15,560 consistently detectable genes during 1, 2, 4, 8, 24, and 96 h of serum starvation (SS), 1, 2, 4, 8, 24, and 48 h serum restimulation (SR), and 7 and 14 days (each repeated twice) of contact inhibition (CI). Expression in serum starvation and contact inhibition is shown relative to proliferating cells, and expression during serum restimulation is shown relative to 4-day serum-starved cells. Colors are as in Figure 1A. Numerals designate 4 different clusters chosen from the hierarchical clustering tree. Select enriched gene ontology terms for each of the clusters are shown in Additional File 1, Table S2. (B) Volcano plot of the mean projection of the microRNA target genes' log2 expression onto the array's first eigengene (Additional File 1, Figure S3B) on the x-axis versus the log10 P value of the mean projection on the y-axis.

Mentions: In order to identify microRNAs with a functional, regulatory role in quiescence, we analyzed the gene expression patterns of microRNA target genes in two whole-genome mRNA microarray timecourses comparing proliferating cells to cells induced into quiescence by contact inhibition or serum starvation (Figure 2A). In one timecourse, fibroblasts were made quiescent by serum withdrawal for 4 days and then re-stimulated with serum for 48 h [54]. In another, fibroblasts were sampled after 7 or 14 days of contact inhibition [52]. Using singular value decomposition of the combined timecourses, we found that the strongest orthonormal gene expression pattern ('eigengene') correlated with the proliferative state of the cell (Additional File 1, Figure S3B). This eigengene explained approximately 40% of the gene expression variation (Additional File 1, Figure S3A). The linear projection of each gene to that eigengene gave a 'proliferation index' for each gene that summarized its association with proliferation or quiescence. For each microRNA, we averaged the proliferation indexes of its predicted target genes as provided by the TargetScan algorithm [55,56] and assigned a P value to that mean using bootstrap resampling (Figure 2B). The miR-29 family's targets had the most statistically extreme mean proliferation index, with a P value <10-4 (the lowest P value possible based on the 104 bootstrap resamplings taken). miR-29 expression is strongly associated with proliferation (Additional File 1, Figure S4), and its predicted targets are upregulated by both methods of quiescence induction.


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)

Changes in target genes with quiescence. (A) Hierarchical clustered heat map representing the log2 fold change of gene expression for all 15,560 consistently detectable genes during 1, 2, 4, 8, 24, and 96 h of serum starvation (SS), 1, 2, 4, 8, 24, and 48 h serum restimulation (SR), and 7 and 14 days (each repeated twice) of contact inhibition (CI). Expression in serum starvation and contact inhibition is shown relative to proliferating cells, and expression during serum restimulation is shown relative to 4-day serum-starved cells. Colors are as in Figure 1A. Numerals designate 4 different clusters chosen from the hierarchical clustering tree. Select enriched gene ontology terms for each of the clusters are shown in Additional File 1, Table S2. (B) Volcano plot of the mean projection of the microRNA target genes' log2 expression onto the array's first eigengene (Additional File 1, Figure S3B) on the x-axis versus the log10 P value of the mean projection on the y-axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Changes in target genes with quiescence. (A) Hierarchical clustered heat map representing the log2 fold change of gene expression for all 15,560 consistently detectable genes during 1, 2, 4, 8, 24, and 96 h of serum starvation (SS), 1, 2, 4, 8, 24, and 48 h serum restimulation (SR), and 7 and 14 days (each repeated twice) of contact inhibition (CI). Expression in serum starvation and contact inhibition is shown relative to proliferating cells, and expression during serum restimulation is shown relative to 4-day serum-starved cells. Colors are as in Figure 1A. Numerals designate 4 different clusters chosen from the hierarchical clustering tree. Select enriched gene ontology terms for each of the clusters are shown in Additional File 1, Table S2. (B) Volcano plot of the mean projection of the microRNA target genes' log2 expression onto the array's first eigengene (Additional File 1, Figure S3B) on the x-axis versus the log10 P value of the mean projection on the y-axis.
Mentions: In order to identify microRNAs with a functional, regulatory role in quiescence, we analyzed the gene expression patterns of microRNA target genes in two whole-genome mRNA microarray timecourses comparing proliferating cells to cells induced into quiescence by contact inhibition or serum starvation (Figure 2A). In one timecourse, fibroblasts were made quiescent by serum withdrawal for 4 days and then re-stimulated with serum for 48 h [54]. In another, fibroblasts were sampled after 7 or 14 days of contact inhibition [52]. Using singular value decomposition of the combined timecourses, we found that the strongest orthonormal gene expression pattern ('eigengene') correlated with the proliferative state of the cell (Additional File 1, Figure S3B). This eigengene explained approximately 40% of the gene expression variation (Additional File 1, Figure S3A). The linear projection of each gene to that eigengene gave a 'proliferation index' for each gene that summarized its association with proliferation or quiescence. For each microRNA, we averaged the proliferation indexes of its predicted target genes as provided by the TargetScan algorithm [55,56] and assigned a P value to that mean using bootstrap resampling (Figure 2B). The miR-29 family's targets had the most statistically extreme mean proliferation index, with a P value <10-4 (the lowest P value possible based on the 104 bootstrap resamplings taken). miR-29 expression is strongly associated with proliferation (Additional File 1, Figure S4), and its predicted targets are upregulated by both methods of quiescence induction.

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