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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.

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.

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miR-29 repression of extracellular matrix protein production with quiescence. (A) Gene expression changes induced 48 h after miR-29 transfection into fibroblasts. The x-axis denotes the mean log2 fold change in expression compared to negative control, and the y-axis denotes -log10 of the P value of a one-sided t-test. (B) Empirical cumulative distribution function of log2 fold-changes induced by miR-29 transfection, comparing predicted targets to all other non-target genes. (C) Quiescence microarray expression timecourses (Figure 2A) of each miR-29 target in Table 1 (shown in gray), along with the mean log2 fold change at each timepoint (shown in red). (D) Protein expression, as determined by immunoblotting, of selected miR-29 targets in proliferating (P), mitogen-starved (MS), or contact inhibited (CI) states with transfection of a negative control microRNA or miR-29. Collagen III here appears as a doublet corresponding to its two isomers. Immunoblots to GAPDH and α-Tubulin are shown as examples of genes not targeted by miR-29 and as loading controls.
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Figure 3: miR-29 repression of extracellular matrix protein production with quiescence. (A) Gene expression changes induced 48 h after miR-29 transfection into fibroblasts. The x-axis denotes the mean log2 fold change in expression compared to negative control, and the y-axis denotes -log10 of the P value of a one-sided t-test. (B) Empirical cumulative distribution function of log2 fold-changes induced by miR-29 transfection, comparing predicted targets to all other non-target genes. (C) Quiescence microarray expression timecourses (Figure 2A) of each miR-29 target in Table 1 (shown in gray), along with the mean log2 fold change at each timepoint (shown in red). (D) Protein expression, as determined by immunoblotting, of selected miR-29 targets in proliferating (P), mitogen-starved (MS), or contact inhibited (CI) states with transfection of a negative control microRNA or miR-29. Collagen III here appears as a doublet corresponding to its two isomers. Immunoblots to GAPDH and α-Tubulin are shown as examples of genes not targeted by miR-29 and as loading controls.

Mentions: Gene ontology analysis of predicted, evolutionarily conserved miR-29 targets revealed an enrichment for multiple categories including collagen fibril organization and extracellular matrix formation (Additional File 1, Table S3), indicating that miR-29 most likely regulates extracellular matrix (ECM) biosynthesis in fibroblasts, consistent with previous reports on miR-29 in fibroblasts and other cell types [67-72]. We identified miR-29 targets in dermal fibroblasts by overexpressing miR-29 in asynchronously proliferating fibroblasts and analyzing the ensuing changes in gene expression by microarray analysis. As expected, genes predicted to be miR-29 targets by TargetScan were more likely to be repressed by miR-29 overexpression than genes not predicted to be miR-29 targets (Figure 3B). We identified genes that both changed significantly in the microarray analysis and contained predicted miR-29 binding sites. Of the 15 genes that met these criteria, nine are involved in extracellular matrix formation (Figure 3A and Table 1). When we plotted the behavior of these same genes in the serum starvation and contact inhibition microarray timecourse data, we discovered that these genes display a quiescence-associated gene expression pattern. The genes encoding miR-29 targets followed a general pattern of increasing expression as fibroblasts are serum-starved, decreasing expression as they are restimulated, and highest expression in cells that were contact-inhibited for 7 or 14 days (Figure 3C). These genes were therefore highly anti-correlated with the pattern of expression for miR-29 itself (Additional File 1, Figure S4). These results suggest that the downregulation of miR-29 expression levels in quiescent fibroblasts is an important contributor to the induction of extracellular matrix genes with quiescence.


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)

miR-29 repression of extracellular matrix protein production with quiescence. (A) Gene expression changes induced 48 h after miR-29 transfection into fibroblasts. The x-axis denotes the mean log2 fold change in expression compared to negative control, and the y-axis denotes -log10 of the P value of a one-sided t-test. (B) Empirical cumulative distribution function of log2 fold-changes induced by miR-29 transfection, comparing predicted targets to all other non-target genes. (C) Quiescence microarray expression timecourses (Figure 2A) of each miR-29 target in Table 1 (shown in gray), along with the mean log2 fold change at each timepoint (shown in red). (D) Protein expression, as determined by immunoblotting, of selected miR-29 targets in proliferating (P), mitogen-starved (MS), or contact inhibited (CI) states with transfection of a negative control microRNA or miR-29. Collagen III here appears as a doublet corresponding to its two isomers. Immunoblots to GAPDH and α-Tubulin are shown as examples of genes not targeted by miR-29 and as loading controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: miR-29 repression of extracellular matrix protein production with quiescence. (A) Gene expression changes induced 48 h after miR-29 transfection into fibroblasts. The x-axis denotes the mean log2 fold change in expression compared to negative control, and the y-axis denotes -log10 of the P value of a one-sided t-test. (B) Empirical cumulative distribution function of log2 fold-changes induced by miR-29 transfection, comparing predicted targets to all other non-target genes. (C) Quiescence microarray expression timecourses (Figure 2A) of each miR-29 target in Table 1 (shown in gray), along with the mean log2 fold change at each timepoint (shown in red). (D) Protein expression, as determined by immunoblotting, of selected miR-29 targets in proliferating (P), mitogen-starved (MS), or contact inhibited (CI) states with transfection of a negative control microRNA or miR-29. Collagen III here appears as a doublet corresponding to its two isomers. Immunoblots to GAPDH and α-Tubulin are shown as examples of genes not targeted by miR-29 and as loading controls.
Mentions: Gene ontology analysis of predicted, evolutionarily conserved miR-29 targets revealed an enrichment for multiple categories including collagen fibril organization and extracellular matrix formation (Additional File 1, Table S3), indicating that miR-29 most likely regulates extracellular matrix (ECM) biosynthesis in fibroblasts, consistent with previous reports on miR-29 in fibroblasts and other cell types [67-72]. We identified miR-29 targets in dermal fibroblasts by overexpressing miR-29 in asynchronously proliferating fibroblasts and analyzing the ensuing changes in gene expression by microarray analysis. As expected, genes predicted to be miR-29 targets by TargetScan were more likely to be repressed by miR-29 overexpression than genes not predicted to be miR-29 targets (Figure 3B). We identified genes that both changed significantly in the microarray analysis and contained predicted miR-29 binding sites. Of the 15 genes that met these criteria, nine are involved in extracellular matrix formation (Figure 3A and Table 1). When we plotted the behavior of these same genes in the serum starvation and contact inhibition microarray timecourse data, we discovered that these genes display a quiescence-associated gene expression pattern. The genes encoding miR-29 targets followed a general pattern of increasing expression as fibroblasts are serum-starved, decreasing expression as they are restimulated, and highest expression in cells that were contact-inhibited for 7 or 14 days (Figure 3C). These genes were therefore highly anti-correlated with the pattern of expression for miR-29 itself (Additional File 1, Figure S4). These results suggest that the downregulation of miR-29 expression levels in quiescent fibroblasts is an important contributor to the induction of extracellular matrix genes with quiescence.

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