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Identification of Spinal Cord MicroRNA and Gene Signatures in a Model of Chronic Stress-Induced Visceral Hyperalgesia in Rat.

Bradesi S, Karagiannides I, Bakirtzi K, Joshi SM, Koukos G, Iliopoulos D, Pothoulakis C, Mayer EA - PLoS ONE (2015)

Bottom Line: In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p.A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

View Article: PubMed Central - PubMed

Affiliation: Oppenheimer Family Center for Neurobiology of Stress, Division of Digestive Diseases, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America; CURE Center, Veterans Administration, Greater Los Angeles, California, United States of America.

ABSTRACT

Introduction: Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.

Methods: Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.

Results: Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.

Conclusion: Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

No MeSH data available.


Real Time RT-PCR verification of selected gene expression after chronic WA stress.IL6ST was significantly up-regulated in stress samples compared to controls while both STAT3 and GFAP were significantly reduced. P<0.5, n = 4 in each group.
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pone.0130938.g004: Real Time RT-PCR verification of selected gene expression after chronic WA stress.IL6ST was significantly up-regulated in stress samples compared to controls while both STAT3 and GFAP were significantly reduced. P<0.5, n = 4 in each group.

Mentions: To confirm changes in mRNA and miRNA expression level observed from our arrays, q-RT-PCR was performed in duplicates from the same RNA samples used for microarray and additional samples from the same experiment. Q-RT-PCR was performed on selected genes and miRNA that were identified in networks and pathways of interest described above. We measured the expression of the Interleukin 6 signal transducer gp130 (IL-6ST), I kappa b kinase epsilon (IKBKE), GFAP, high mobility group box 1 (Hmbg1), signal transducer and activator of transcription 3 (acute-phase response factor (STAT3) and mir17-5p, 148-3p and 19b-3p. We observed a significant increase in IL6ST (gp130) in samples from stressed rats compared to controls (Fig 4). STAT3 and GFAP expression were significantly decreased in stressed rats compared to controls (Fig 4). While there was a trend for an increased expression of IKBKE and Hmbg1 in stressed rats compared to control, the changes were not significant (Fig 4). We also found a significant increase in the expression of mir17-5p in stressed rats compared to controls. In contrast, there was no change in the expression of mir148-3p or mir19b-3p (Fig 5).


Identification of Spinal Cord MicroRNA and Gene Signatures in a Model of Chronic Stress-Induced Visceral Hyperalgesia in Rat.

Bradesi S, Karagiannides I, Bakirtzi K, Joshi SM, Koukos G, Iliopoulos D, Pothoulakis C, Mayer EA - PLoS ONE (2015)

Real Time RT-PCR verification of selected gene expression after chronic WA stress.IL6ST was significantly up-regulated in stress samples compared to controls while both STAT3 and GFAP were significantly reduced. P<0.5, n = 4 in each group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519289&req=5

pone.0130938.g004: Real Time RT-PCR verification of selected gene expression after chronic WA stress.IL6ST was significantly up-regulated in stress samples compared to controls while both STAT3 and GFAP were significantly reduced. P<0.5, n = 4 in each group.
Mentions: To confirm changes in mRNA and miRNA expression level observed from our arrays, q-RT-PCR was performed in duplicates from the same RNA samples used for microarray and additional samples from the same experiment. Q-RT-PCR was performed on selected genes and miRNA that were identified in networks and pathways of interest described above. We measured the expression of the Interleukin 6 signal transducer gp130 (IL-6ST), I kappa b kinase epsilon (IKBKE), GFAP, high mobility group box 1 (Hmbg1), signal transducer and activator of transcription 3 (acute-phase response factor (STAT3) and mir17-5p, 148-3p and 19b-3p. We observed a significant increase in IL6ST (gp130) in samples from stressed rats compared to controls (Fig 4). STAT3 and GFAP expression were significantly decreased in stressed rats compared to controls (Fig 4). While there was a trend for an increased expression of IKBKE and Hmbg1 in stressed rats compared to control, the changes were not significant (Fig 4). We also found a significant increase in the expression of mir17-5p in stressed rats compared to controls. In contrast, there was no change in the expression of mir148-3p or mir19b-3p (Fig 5).

Bottom Line: In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p.A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

View Article: PubMed Central - PubMed

Affiliation: Oppenheimer Family Center for Neurobiology of Stress, Division of Digestive Diseases, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America; CURE Center, Veterans Administration, Greater Los Angeles, California, United States of America.

ABSTRACT

Introduction: Animal studies have shown that stress could induce epigenetic and transcriptomic alterations essential in determining the balance between adaptive or maladaptive responses to stress. We tested the hypothesis that chronic stress in rats deregulates coding and non-coding gene expression in the spinal cord, which may underline neuroinflammation and nociceptive changes previously observed in this model.

Methods: Male Wistar rats were exposed to daily stress or handled, for 10 days. At day 11, lumbar spinal segments were collected and processed for mRNA/miRNA isolation followed by expression profiling using Agilent SurePrint Rat Exon and Rat miRNA Microarray platforms. Differentially expressed gene lists were generated using the dChip program. Microarrays were analyzed using the Ingenuity Pathways Analysis (IPA) tool from Ingenuity Systems. Multiple methods were used for the analysis of miRNA-mRNA functional modules. Quantitative real time RT-PCR for Interleukin 6 signal transducer (gp130), the Signal Transducer And Activator Of Transcription 3 (STAT3), glial fibrillary acidic protein and mir-17-5p were performed to confirm levels of expression.

Results: Gene network analysis revealed that stress deregulated different inflammatory (IL-6, JAK/STAT, TNF) and metabolic (PI3K/AKT) signaling pathways. MicroRNA array analysis revealed a signature of 39 deregulated microRNAs in stressed rats. MicroRNA-gene network analysis showed that microRNAs are regulators of two gene networks relevant to inflammatory processes. Specifically, our analysis of miRNA-mRNA functional modules identified miR-17-5p as an important regulator in our model. We verified miR-17-5p increased expression in stress using qPCR and in situ hybridization. In addition, we observed changes in the expression of gp130 and STAT3 (involved in intracellular signaling cascades in response to gp130 activation), both predicted targets for miR-17-5p. A modulatory role of spinal mir17-5p in the modulation of visceral sensitivity was confirmed in vivo.

Conclusion: Using an integrative high throughput approach, our findings suggest a link between miR-17-5p increased expression and gp130/STAT3 activation providing new insight into the possible mechanisms mediating the effect of chronic stress on neuroinflammation in the spinal cord.

No MeSH data available.