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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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Validation of novel glucocorticoid responsive miRNAs.(A) Secondary structure of two novel miRNA generated by ViennaRNA. The predicted ‘mature’ sequence is highlighted in red; the remaining hairpin contains the putative stem loop and mature-star* sequence, the minimum free energy (MFE) of each structure is indicated. The VARNA visualization applet was used to draw the RNA secondary structure [66].(B) The expression of the candidate novel miRNAs, candidates 44 and 166 visualized in UCSC genome browser (Dex treated is top bar, Control is bottom bar). Both of the predicted novel miRNAs are repressed during glucocorticoid-induced apoptosis of primary lymphocytes. Visualization of novel miRNA candidate 166 also detects a glucocorticoid-responsive signal at the proximal newly annotated mature miRNA rno-miR-6324, which is antisense to candidate 166 (indicated in the red box).(C) Percent control values of the five miRNAs (3 known and 2 predicted novel candidates) selected for qPCR validation. Percent control was calculated as (Dex/Control) using computationally derived signal values for control and dexamethasone-treated rat primary thymocytes. Signal values were generated using stringent sequence alignment criteria of miRNA-seq data. (D) Graphic representation of percent control values for control and dexamethasone-treated samples generated using computationally derived expression signals from the miRNA-seq data. Raw read counts at each miRNA were normalized to the total number of aligned reads in the respective sample to generate normalized signal. (E) Rat primary thymocytes were untreated (control) or treated with 100nM dexamethasone for 6 hours (apoptosis was monitored as previously described [1]). The expression of annotated positive controls and individual mature candidates was evaluated via quantitative PCR using custom TaqMan Small RNA Assays. The expression of RNU43 small nuclear RNA served as an endogenous control. Results are reported as mean percent control values +/- SEM values for 3 biological replicates (**p<.01).
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pone-0078316-g002: Validation of novel glucocorticoid responsive miRNAs.(A) Secondary structure of two novel miRNA generated by ViennaRNA. The predicted ‘mature’ sequence is highlighted in red; the remaining hairpin contains the putative stem loop and mature-star* sequence, the minimum free energy (MFE) of each structure is indicated. The VARNA visualization applet was used to draw the RNA secondary structure [66].(B) The expression of the candidate novel miRNAs, candidates 44 and 166 visualized in UCSC genome browser (Dex treated is top bar, Control is bottom bar). Both of the predicted novel miRNAs are repressed during glucocorticoid-induced apoptosis of primary lymphocytes. Visualization of novel miRNA candidate 166 also detects a glucocorticoid-responsive signal at the proximal newly annotated mature miRNA rno-miR-6324, which is antisense to candidate 166 (indicated in the red box).(C) Percent control values of the five miRNAs (3 known and 2 predicted novel candidates) selected for qPCR validation. Percent control was calculated as (Dex/Control) using computationally derived signal values for control and dexamethasone-treated rat primary thymocytes. Signal values were generated using stringent sequence alignment criteria of miRNA-seq data. (D) Graphic representation of percent control values for control and dexamethasone-treated samples generated using computationally derived expression signals from the miRNA-seq data. Raw read counts at each miRNA were normalized to the total number of aligned reads in the respective sample to generate normalized signal. (E) Rat primary thymocytes were untreated (control) or treated with 100nM dexamethasone for 6 hours (apoptosis was monitored as previously described [1]). The expression of annotated positive controls and individual mature candidates was evaluated via quantitative PCR using custom TaqMan Small RNA Assays. The expression of RNU43 small nuclear RNA served as an endogenous control. Results are reported as mean percent control values +/- SEM values for 3 biological replicates (**p<.01).

Mentions: To verify the glucocorticoid-induced repression of miRNAs, a combination of both annotated and novel miRNA candidates were selected for qPCR validation. Two novel miRNA candidates, candidate 44 and candidate 166, were chosen for validation on the basis of their predicted secondary structure. Both candidates demonstrate a canonical stem-loop structure and a putative mature miRNA sequence (Figure 2A). Furthermore, the expression of each novel miRNA candidate (as visualized in the UCSC Genome Browser [26]) is repressed in response to dexamethasone treatment (Figure 2B). This observation parallels the trend of prevalent repression of annotated miRNAs during glucocorticoid-induced apoptosis of lymphocytes, suggesting that these novel candidates are biologically similar to annotated miRNAs. Interestingly, candidate 166 also exhibits detectable signal at the proximal mature miRNA rno-miR-6324, a recently annotated mature miRNA arising from the same precursor as candidate 166 [27]. While the basal expression of rno-miR-3624 is lower than candidate 166, it is also repressed in response to dexamethasone treatment (Figure 2B).


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)

Validation of novel glucocorticoid responsive miRNAs.(A) Secondary structure of two novel miRNA generated by ViennaRNA. The predicted ‘mature’ sequence is highlighted in red; the remaining hairpin contains the putative stem loop and mature-star* sequence, the minimum free energy (MFE) of each structure is indicated. The VARNA visualization applet was used to draw the RNA secondary structure [66].(B) The expression of the candidate novel miRNAs, candidates 44 and 166 visualized in UCSC genome browser (Dex treated is top bar, Control is bottom bar). Both of the predicted novel miRNAs are repressed during glucocorticoid-induced apoptosis of primary lymphocytes. Visualization of novel miRNA candidate 166 also detects a glucocorticoid-responsive signal at the proximal newly annotated mature miRNA rno-miR-6324, which is antisense to candidate 166 (indicated in the red box).(C) Percent control values of the five miRNAs (3 known and 2 predicted novel candidates) selected for qPCR validation. Percent control was calculated as (Dex/Control) using computationally derived signal values for control and dexamethasone-treated rat primary thymocytes. Signal values were generated using stringent sequence alignment criteria of miRNA-seq data. (D) Graphic representation of percent control values for control and dexamethasone-treated samples generated using computationally derived expression signals from the miRNA-seq data. Raw read counts at each miRNA were normalized to the total number of aligned reads in the respective sample to generate normalized signal. (E) Rat primary thymocytes were untreated (control) or treated with 100nM dexamethasone for 6 hours (apoptosis was monitored as previously described [1]). The expression of annotated positive controls and individual mature candidates was evaluated via quantitative PCR using custom TaqMan Small RNA Assays. The expression of RNU43 small nuclear RNA served as an endogenous control. Results are reported as mean percent control values +/- SEM values for 3 biological replicates (**p<.01).
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pone-0078316-g002: Validation of novel glucocorticoid responsive miRNAs.(A) Secondary structure of two novel miRNA generated by ViennaRNA. The predicted ‘mature’ sequence is highlighted in red; the remaining hairpin contains the putative stem loop and mature-star* sequence, the minimum free energy (MFE) of each structure is indicated. The VARNA visualization applet was used to draw the RNA secondary structure [66].(B) The expression of the candidate novel miRNAs, candidates 44 and 166 visualized in UCSC genome browser (Dex treated is top bar, Control is bottom bar). Both of the predicted novel miRNAs are repressed during glucocorticoid-induced apoptosis of primary lymphocytes. Visualization of novel miRNA candidate 166 also detects a glucocorticoid-responsive signal at the proximal newly annotated mature miRNA rno-miR-6324, which is antisense to candidate 166 (indicated in the red box).(C) Percent control values of the five miRNAs (3 known and 2 predicted novel candidates) selected for qPCR validation. Percent control was calculated as (Dex/Control) using computationally derived signal values for control and dexamethasone-treated rat primary thymocytes. Signal values were generated using stringent sequence alignment criteria of miRNA-seq data. (D) Graphic representation of percent control values for control and dexamethasone-treated samples generated using computationally derived expression signals from the miRNA-seq data. Raw read counts at each miRNA were normalized to the total number of aligned reads in the respective sample to generate normalized signal. (E) Rat primary thymocytes were untreated (control) or treated with 100nM dexamethasone for 6 hours (apoptosis was monitored as previously described [1]). The expression of annotated positive controls and individual mature candidates was evaluated via quantitative PCR using custom TaqMan Small RNA Assays. The expression of RNU43 small nuclear RNA served as an endogenous control. Results are reported as mean percent control values +/- SEM values for 3 biological replicates (**p<.01).
Mentions: To verify the glucocorticoid-induced repression of miRNAs, a combination of both annotated and novel miRNA candidates were selected for qPCR validation. Two novel miRNA candidates, candidate 44 and candidate 166, were chosen for validation on the basis of their predicted secondary structure. Both candidates demonstrate a canonical stem-loop structure and a putative mature miRNA sequence (Figure 2A). Furthermore, the expression of each novel miRNA candidate (as visualized in the UCSC Genome Browser [26]) is repressed in response to dexamethasone treatment (Figure 2B). This observation parallels the trend of prevalent repression of annotated miRNAs during glucocorticoid-induced apoptosis of lymphocytes, suggesting that these novel candidates are biologically similar to annotated miRNAs. Interestingly, candidate 166 also exhibits detectable signal at the proximal mature miRNA rno-miR-6324, a recently annotated mature miRNA arising from the same precursor as candidate 166 [27]. While the basal expression of rno-miR-3624 is lower than candidate 166, it is also repressed in response to dexamethasone treatment (Figure 2B).

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