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Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts.

Greussing R, Hackl M, Charoentong P, Pauck A, Monteforte R, Cavinato M, Hofer E, Scheideler M, Neuhaus M, Micutkova L, Mueck C, Trajanoski Z, Grillari J, Jansen-Dürr P - BMC Genomics (2013)

Bottom Line: In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process.We performed a comprehensive screen for UVB-regulated microRNAs in human diploid fibroblasts, and identified a network of miRNA-mRNA interactions mediating UVB-induced senescence.In addition, miR-101 and Ezh2 were identified as key players in UVB-induced senescence of HDF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, Innsbruck 6020, Austria. Pidder.Jansen-Duerr@uibk.ac.at.

ABSTRACT

Background: Cellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts.

Results: We observed a parallel activation of the p53/p21(WAF1) and p16(INK4a)/pRb pathways. Using genome-wide transcriptome analysis, we identified a transcriptional signature of UVB-induced senescence that was conserved in three independent strains of human diploid fibroblasts (HDF) from skin. In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process. Bioinformatic analysis of miRNA-mRNA networks was performed to identify new functional mRNA targets with high confidence for miR-15a, miR-20a, miR-20b, miR-93, and miR-101. Already known targets of these miRNAs were identified in each case, validating the approach. Several new targets were identified for all of these miRNAs, with the potential to provide new insight in the process of UVB-induced senescence at a genome-wide level. Subsequent analysis was focused on miR-101 and its putative target gene Ezh2. We confirmed that Ezh2 is regulated by miR-101 in human fibroblasts, and found that both overexpression of miR-101 and downregulation of Ezh2 independently induce senescence in the absence of UVB irradiation. However, the downregulation of miR-101 was not sufficient to block the phenotype of UVB-induced senescence, suggesting that other UVB-induced processes induce the senescence response in a pathway redundant with upregulation of miR-101.

Conclusion: We performed a comprehensive screen for UVB-regulated microRNAs in human diploid fibroblasts, and identified a network of miRNA-mRNA interactions mediating UVB-induced senescence. In addition, miR-101 and Ezh2 were identified as key players in UVB-induced senescence of HDF.

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Depletion of miR-101 fails to prevent UVB-induced senescence. Cells were irradiated with UVB twice a day for 4 days. After the last exposure the cells were reverse transfected as described with miR-101 power inhibitors for knockdown, negative control or not treated. Reverse transfection was repeated at day 7. At day 9 RNA and protein were isolated. A. Control cells. Expression levels of miR-101 and Ezh2 mRNA of non-irradiated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. B. UVB-treated cells miR-101 and Ezh2 expression levels from UVB treated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. C. Growth curve analysis of miR-101 knockdown cells. cPDLs were calculated as described. Data represent the mean ± SD of three independent experiments. cPDL: cumulative population doublings KD: knockdown. *p < 0.01; **p < 0.001.
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Figure 8: Depletion of miR-101 fails to prevent UVB-induced senescence. Cells were irradiated with UVB twice a day for 4 days. After the last exposure the cells were reverse transfected as described with miR-101 power inhibitors for knockdown, negative control or not treated. Reverse transfection was repeated at day 7. At day 9 RNA and protein were isolated. A. Control cells. Expression levels of miR-101 and Ezh2 mRNA of non-irradiated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. B. UVB-treated cells miR-101 and Ezh2 expression levels from UVB treated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. C. Growth curve analysis of miR-101 knockdown cells. cPDLs were calculated as described. Data represent the mean ± SD of three independent experiments. cPDL: cumulative population doublings KD: knockdown. *p < 0.01; **p < 0.001.

Mentions: To address the role of miR-101 in UVB-induced cellular senescence, we attempted to experimentally reduce miR-101 levels by transfection of miR-101 inhibitory RNAs. This treatment was started one day before UVB treatment and continued thereafter, in order to keep miR-101 levels constantly low. Using reverse transfection, the levels of miR-101 were significantly reduced in control HDF (Figure 8A) and miR-101 knockdown efficiently abrogated miR-101 upregulation in UVB-treated fibroblasts (Figure 8B). However, Ezh2 mRNA was not significantly upregulated in miR-101 depleted cells, irrespective of UVB treatment (Figure 8A,B); and miR-101 knockdown failed to increase Ezh2 protein levels in both cases (data not shown). To address potential mechanisms underlying the failure to upregulate Ezh2, Ezh2 was also overexpressed by lentiviral vectors. Both in HDF and in easy-to-transfect human osteosarcoma (U2-OS) cells, overexpression of Ezh2 mRNA was transient and no elevation of Ezh2 protein levels was observed in HDF (Additional file 8: Figure S6), for reasons that remain to be established. Together, these experiments indicate that reduction of miR-101 was not sufficient to rescue Ezh2 expression in UVB-treated cells. Accordingly, the phenotype of UVB-induced cellular senescence was not significantly affected by preventing the UVB-induced upregulation of miR-101 (Figure 8C). Consistent with this observation, knockdown of miR-101 also failed to significantly reduce the percentage of SA-β-gal positive cells after UVB treatment (data not shown).


Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts.

Greussing R, Hackl M, Charoentong P, Pauck A, Monteforte R, Cavinato M, Hofer E, Scheideler M, Neuhaus M, Micutkova L, Mueck C, Trajanoski Z, Grillari J, Jansen-Dürr P - BMC Genomics (2013)

Depletion of miR-101 fails to prevent UVB-induced senescence. Cells were irradiated with UVB twice a day for 4 days. After the last exposure the cells were reverse transfected as described with miR-101 power inhibitors for knockdown, negative control or not treated. Reverse transfection was repeated at day 7. At day 9 RNA and protein were isolated. A. Control cells. Expression levels of miR-101 and Ezh2 mRNA of non-irradiated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. B. UVB-treated cells miR-101 and Ezh2 expression levels from UVB treated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. C. Growth curve analysis of miR-101 knockdown cells. cPDLs were calculated as described. Data represent the mean ± SD of three independent experiments. cPDL: cumulative population doublings KD: knockdown. *p < 0.01; **p < 0.001.
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Figure 8: Depletion of miR-101 fails to prevent UVB-induced senescence. Cells were irradiated with UVB twice a day for 4 days. After the last exposure the cells were reverse transfected as described with miR-101 power inhibitors for knockdown, negative control or not treated. Reverse transfection was repeated at day 7. At day 9 RNA and protein were isolated. A. Control cells. Expression levels of miR-101 and Ezh2 mRNA of non-irradiated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. B. UVB-treated cells miR-101 and Ezh2 expression levels from UVB treated cells after miR-101 knockdown were determined by real-time qPCR. Bars indicate the mean ± SE of three independent experiments. C. Growth curve analysis of miR-101 knockdown cells. cPDLs were calculated as described. Data represent the mean ± SD of three independent experiments. cPDL: cumulative population doublings KD: knockdown. *p < 0.01; **p < 0.001.
Mentions: To address the role of miR-101 in UVB-induced cellular senescence, we attempted to experimentally reduce miR-101 levels by transfection of miR-101 inhibitory RNAs. This treatment was started one day before UVB treatment and continued thereafter, in order to keep miR-101 levels constantly low. Using reverse transfection, the levels of miR-101 were significantly reduced in control HDF (Figure 8A) and miR-101 knockdown efficiently abrogated miR-101 upregulation in UVB-treated fibroblasts (Figure 8B). However, Ezh2 mRNA was not significantly upregulated in miR-101 depleted cells, irrespective of UVB treatment (Figure 8A,B); and miR-101 knockdown failed to increase Ezh2 protein levels in both cases (data not shown). To address potential mechanisms underlying the failure to upregulate Ezh2, Ezh2 was also overexpressed by lentiviral vectors. Both in HDF and in easy-to-transfect human osteosarcoma (U2-OS) cells, overexpression of Ezh2 mRNA was transient and no elevation of Ezh2 protein levels was observed in HDF (Additional file 8: Figure S6), for reasons that remain to be established. Together, these experiments indicate that reduction of miR-101 was not sufficient to rescue Ezh2 expression in UVB-treated cells. Accordingly, the phenotype of UVB-induced cellular senescence was not significantly affected by preventing the UVB-induced upregulation of miR-101 (Figure 8C). Consistent with this observation, knockdown of miR-101 also failed to significantly reduce the percentage of SA-β-gal positive cells after UVB treatment (data not shown).

Bottom Line: In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process.We performed a comprehensive screen for UVB-regulated microRNAs in human diploid fibroblasts, and identified a network of miRNA-mRNA interactions mediating UVB-induced senescence.In addition, miR-101 and Ezh2 were identified as key players in UVB-induced senescence of HDF.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, Innsbruck 6020, Austria. Pidder.Jansen-Duerr@uibk.ac.at.

ABSTRACT

Background: Cellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts.

Results: We observed a parallel activation of the p53/p21(WAF1) and p16(INK4a)/pRb pathways. Using genome-wide transcriptome analysis, we identified a transcriptional signature of UVB-induced senescence that was conserved in three independent strains of human diploid fibroblasts (HDF) from skin. In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process. Bioinformatic analysis of miRNA-mRNA networks was performed to identify new functional mRNA targets with high confidence for miR-15a, miR-20a, miR-20b, miR-93, and miR-101. Already known targets of these miRNAs were identified in each case, validating the approach. Several new targets were identified for all of these miRNAs, with the potential to provide new insight in the process of UVB-induced senescence at a genome-wide level. Subsequent analysis was focused on miR-101 and its putative target gene Ezh2. We confirmed that Ezh2 is regulated by miR-101 in human fibroblasts, and found that both overexpression of miR-101 and downregulation of Ezh2 independently induce senescence in the absence of UVB irradiation. However, the downregulation of miR-101 was not sufficient to block the phenotype of UVB-induced senescence, suggesting that other UVB-induced processes induce the senescence response in a pathway redundant with upregulation of miR-101.

Conclusion: We performed a comprehensive screen for UVB-regulated microRNAs in human diploid fibroblasts, and identified a network of miRNA-mRNA interactions mediating UVB-induced senescence. In addition, miR-101 and Ezh2 were identified as key players in UVB-induced senescence of HDF.

Show MeSH
Related in: MedlinePlus