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Deep sequencing identification of novel glucocorticoid-responsive miRNAs in apoptotic primary lymphocytes.

Smith LK, Tandon A, Shah RR, Mav D, Scoltock AB, Cidlowski JA - PLoS ONE (2013)

Bottom Line: This analysis identifies the potential presence of over 200 novel glucocorticoid-responsive miRNAs.We have validated the expression of two novel glucocorticoid-responsive miRNAs using small RNA-specific qPCR.Furthermore, through the use of Ingenuity Pathways Analysis (IPA) we determined that the putative targets of these novel validated miRNAs are predicted to regulate cell death processes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, NIH, Department of Health and Human Services, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
Apoptosis of lymphocytes governs the response of the immune system to environmental stress and toxic insult. Signaling through the ubiquitously expressed glucocorticoid receptor, stress-induced glucocorticoid hormones induce apoptosis via mechanisms requiring altered gene expression. Several reports have detailed the changes in gene expression mediating glucocorticoid-induced apoptosis of lymphocytes. However, few studies have examined the role of non-coding miRNAs in this essential physiological process. Previously, using hybridization-based gene expression analysis and deep sequencing of small RNAs, we described the prevalent post-transcriptional repression of annotated miRNAs during glucocorticoid-induced apoptosis of lymphocytes. Here, we describe the development of a customized bioinformatics pipeline that facilitates the deep sequencing-mediated discovery of novel glucocorticoid-responsive miRNAs in apoptotic primary lymphocytes. This analysis identifies the potential presence of over 200 novel glucocorticoid-responsive miRNAs. We have validated the expression of two novel glucocorticoid-responsive miRNAs using small RNA-specific qPCR. Furthermore, through the use of Ingenuity Pathways Analysis (IPA) we determined that the putative targets of these novel validated miRNAs are predicted to regulate cell death processes. These findings identify two and predict the presence of additional novel glucocorticoid-responsive miRNAs in the rat transcriptome, suggesting a potential role for both annotated and novel miRNAs in glucocorticoid-induced apoptosis of lymphocytes.

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Pathways analysis predicts novel miRNA targets may contribute to glucocorticoid-induced apoptosis.(A) miRNA target predictions for novel miRNA candidates 44 and 166 were performed using the miRanda miRNA target prediction algorithm. The number of target mRNAs differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2) is indicated for each candidate. (B) IPA-generated ranking of the top five molecular and cellular functions of genes differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2), as well as the predicted targets of both candidates 44 and 166 (p-values for top functions are indicated beneath each ranking). Genes differentially expressed during glucocorticoid-induced apoptosis were identified by whole genome microarray analysis of untreated and 100nM dexamethasone-treated thymocytes (6 hours, 3 biological replicates). (C) Venn diagram analysis identified specific novel candidate predicted targets differentially expressed during glucocorticoid-induced apoptosis (p<.01) and the application IPA to this combined gene list (40 genes) generated a top 5 ranking of molecular and cellular functions regulated by these predicted targets (p-values for top functions are indicated beneath each ranking).
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pone-0078316-g003: Pathways analysis predicts novel miRNA targets may contribute to glucocorticoid-induced apoptosis.(A) miRNA target predictions for novel miRNA candidates 44 and 166 were performed using the miRanda miRNA target prediction algorithm. The number of target mRNAs differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2) is indicated for each candidate. (B) IPA-generated ranking of the top five molecular and cellular functions of genes differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2), as well as the predicted targets of both candidates 44 and 166 (p-values for top functions are indicated beneath each ranking). Genes differentially expressed during glucocorticoid-induced apoptosis were identified by whole genome microarray analysis of untreated and 100nM dexamethasone-treated thymocytes (6 hours, 3 biological replicates). (C) Venn diagram analysis identified specific novel candidate predicted targets differentially expressed during glucocorticoid-induced apoptosis (p<.01) and the application IPA to this combined gene list (40 genes) generated a top 5 ranking of molecular and cellular functions regulated by these predicted targets (p-values for top functions are indicated beneath each ranking).

Mentions: Using the mature sequence of novel miRNA candidates 44 and 166, gene target predictions were made against the 3’ untranslated regions of RefSeq transcripts via the miRanda miRNA target prediction algorithm [29]. Numerous gene targets were predicted for both candidate novel miRNAs (Figure 3A). To assess the potential role of these predicted targets in the glucocorticoid-induced apoptosis program, whole genome gene expression microarray was performed on both untreated and dexamethasone treated primary thymocytes (3 biological replicates each). Ingenuity Pathways Analysis (IPA) of genes deemed differentially expressed (p-value < 0.01 and absolute fold change > 1.2) suggests that they govern molecular and cellular functions involving cell proliferation, cell division, and cell death (Figure 3B). Interestingly, IPA of the predicted novel miRNA targets suggests that these miRNAs may contribute to many of the same molecular and cellular functions identified by the whole genome microarray analysis. Specifically, cell death and cell survival is a top IPA-generated molecular and cellular function for the miRanda predicted targets of both candidates 44 and 166 (Figure 3B).


Deep sequencing identification of novel glucocorticoid-responsive miRNAs in apoptotic primary lymphocytes.

Smith LK, Tandon A, Shah RR, Mav D, Scoltock AB, Cidlowski JA - PLoS ONE (2013)

Pathways analysis predicts novel miRNA targets may contribute to glucocorticoid-induced apoptosis.(A) miRNA target predictions for novel miRNA candidates 44 and 166 were performed using the miRanda miRNA target prediction algorithm. The number of target mRNAs differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2) is indicated for each candidate. (B) IPA-generated ranking of the top five molecular and cellular functions of genes differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2), as well as the predicted targets of both candidates 44 and 166 (p-values for top functions are indicated beneath each ranking). Genes differentially expressed during glucocorticoid-induced apoptosis were identified by whole genome microarray analysis of untreated and 100nM dexamethasone-treated thymocytes (6 hours, 3 biological replicates). (C) Venn diagram analysis identified specific novel candidate predicted targets differentially expressed during glucocorticoid-induced apoptosis (p<.01) and the application IPA to this combined gene list (40 genes) generated a top 5 ranking of molecular and cellular functions regulated by these predicted targets (p-values for top functions are indicated beneath each ranking).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3824063&req=5

pone-0078316-g003: Pathways analysis predicts novel miRNA targets may contribute to glucocorticoid-induced apoptosis.(A) miRNA target predictions for novel miRNA candidates 44 and 166 were performed using the miRanda miRNA target prediction algorithm. The number of target mRNAs differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2) is indicated for each candidate. (B) IPA-generated ranking of the top five molecular and cellular functions of genes differentially expressed during glucocorticoid-induced apoptosis (p < 0.01; fold change > 1.2), as well as the predicted targets of both candidates 44 and 166 (p-values for top functions are indicated beneath each ranking). Genes differentially expressed during glucocorticoid-induced apoptosis were identified by whole genome microarray analysis of untreated and 100nM dexamethasone-treated thymocytes (6 hours, 3 biological replicates). (C) Venn diagram analysis identified specific novel candidate predicted targets differentially expressed during glucocorticoid-induced apoptosis (p<.01) and the application IPA to this combined gene list (40 genes) generated a top 5 ranking of molecular and cellular functions regulated by these predicted targets (p-values for top functions are indicated beneath each ranking).
Mentions: Using the mature sequence of novel miRNA candidates 44 and 166, gene target predictions were made against the 3’ untranslated regions of RefSeq transcripts via the miRanda miRNA target prediction algorithm [29]. Numerous gene targets were predicted for both candidate novel miRNAs (Figure 3A). To assess the potential role of these predicted targets in the glucocorticoid-induced apoptosis program, whole genome gene expression microarray was performed on both untreated and dexamethasone treated primary thymocytes (3 biological replicates each). Ingenuity Pathways Analysis (IPA) of genes deemed differentially expressed (p-value < 0.01 and absolute fold change > 1.2) suggests that they govern molecular and cellular functions involving cell proliferation, cell division, and cell death (Figure 3B). Interestingly, IPA of the predicted novel miRNA targets suggests that these miRNAs may contribute to many of the same molecular and cellular functions identified by the whole genome microarray analysis. Specifically, cell death and cell survival is a top IPA-generated molecular and cellular function for the miRanda predicted targets of both candidates 44 and 166 (Figure 3B).

Bottom Line: This analysis identifies the potential presence of over 200 novel glucocorticoid-responsive miRNAs.We have validated the expression of two novel glucocorticoid-responsive miRNAs using small RNA-specific qPCR.Furthermore, through the use of Ingenuity Pathways Analysis (IPA) we determined that the putative targets of these novel validated miRNAs are predicted to regulate cell death processes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, NIH, Department of Health and Human Services, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
Apoptosis of lymphocytes governs the response of the immune system to environmental stress and toxic insult. Signaling through the ubiquitously expressed glucocorticoid receptor, stress-induced glucocorticoid hormones induce apoptosis via mechanisms requiring altered gene expression. Several reports have detailed the changes in gene expression mediating glucocorticoid-induced apoptosis of lymphocytes. However, few studies have examined the role of non-coding miRNAs in this essential physiological process. Previously, using hybridization-based gene expression analysis and deep sequencing of small RNAs, we described the prevalent post-transcriptional repression of annotated miRNAs during glucocorticoid-induced apoptosis of lymphocytes. Here, we describe the development of a customized bioinformatics pipeline that facilitates the deep sequencing-mediated discovery of novel glucocorticoid-responsive miRNAs in apoptotic primary lymphocytes. This analysis identifies the potential presence of over 200 novel glucocorticoid-responsive miRNAs. We have validated the expression of two novel glucocorticoid-responsive miRNAs using small RNA-specific qPCR. Furthermore, through the use of Ingenuity Pathways Analysis (IPA) we determined that the putative targets of these novel validated miRNAs are predicted to regulate cell death processes. These findings identify two and predict the presence of additional novel glucocorticoid-responsive miRNAs in the rat transcriptome, suggesting a potential role for both annotated and novel miRNAs in glucocorticoid-induced apoptosis of lymphocytes.

Show MeSH
Related in: MedlinePlus