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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.


Effect of the miRCURY LNA mir17-5p inhibitor or scramble control on stress-induced visceral hyperalgesia.A) EMG response to graded CRD at baseline and 24 hours following the 10th stress session showed overall increase of the EMG response to CRD with significant increased at 40 mmHg consistent with stress-induced visceral hyperalgesia n = 8 *P<0.05 two-way ANOVA followed by Bonferroni post test. B) Stressed rats treated with the LNAmir17-5p inhibitor exhibit increased EMG response to CRD at day 11 compared with baseline indicating visceral hyperalgesia n = 6, *P<0.05 two-way ANOVA followed by Bonferroni post test. C) The mean change from baseline was significantly higher in stressed rats treated with the LNAmir17-5p inhibitor (n = 6) compared with stressed rats treated with the control scramble (n = 8) indicating an exacerbation of visceral sensitivity *P<0.05 two-way ANOVA followed by Bonferroni post test.
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pone.0130938.g008: Effect of the miRCURY LNA mir17-5p inhibitor or scramble control on stress-induced visceral hyperalgesia.A) EMG response to graded CRD at baseline and 24 hours following the 10th stress session showed overall increase of the EMG response to CRD with significant increased at 40 mmHg consistent with stress-induced visceral hyperalgesia n = 8 *P<0.05 two-way ANOVA followed by Bonferroni post test. B) Stressed rats treated with the LNAmir17-5p inhibitor exhibit increased EMG response to CRD at day 11 compared with baseline indicating visceral hyperalgesia n = 6, *P<0.05 two-way ANOVA followed by Bonferroni post test. C) The mean change from baseline was significantly higher in stressed rats treated with the LNAmir17-5p inhibitor (n = 6) compared with stressed rats treated with the control scramble (n = 8) indicating an exacerbation of visceral sensitivity *P<0.05 two-way ANOVA followed by Bonferroni post test.

Mentions: Stressed rats treated with the scramble negative control probe showed significant increase of VMR to CRD at day 11 compared with baseline at 60 mmHg. These results are consistent with our previous report that chronic WA induces visceral hyperalgesia[13]. Interestingly, rats exposed to WA stress and receiving the treatment with the miRCURY LNA mir17-5p inhibitor probe exhibited exacerbated increase of the VMR to CRD compared with the scramble control (Fig 8).


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)

Effect of the miRCURY LNA mir17-5p inhibitor or scramble control on stress-induced visceral hyperalgesia.A) EMG response to graded CRD at baseline and 24 hours following the 10th stress session showed overall increase of the EMG response to CRD with significant increased at 40 mmHg consistent with stress-induced visceral hyperalgesia n = 8 *P<0.05 two-way ANOVA followed by Bonferroni post test. B) Stressed rats treated with the LNAmir17-5p inhibitor exhibit increased EMG response to CRD at day 11 compared with baseline indicating visceral hyperalgesia n = 6, *P<0.05 two-way ANOVA followed by Bonferroni post test. C) The mean change from baseline was significantly higher in stressed rats treated with the LNAmir17-5p inhibitor (n = 6) compared with stressed rats treated with the control scramble (n = 8) indicating an exacerbation of visceral sensitivity *P<0.05 two-way ANOVA followed by Bonferroni post test.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4519289&req=5

pone.0130938.g008: Effect of the miRCURY LNA mir17-5p inhibitor or scramble control on stress-induced visceral hyperalgesia.A) EMG response to graded CRD at baseline and 24 hours following the 10th stress session showed overall increase of the EMG response to CRD with significant increased at 40 mmHg consistent with stress-induced visceral hyperalgesia n = 8 *P<0.05 two-way ANOVA followed by Bonferroni post test. B) Stressed rats treated with the LNAmir17-5p inhibitor exhibit increased EMG response to CRD at day 11 compared with baseline indicating visceral hyperalgesia n = 6, *P<0.05 two-way ANOVA followed by Bonferroni post test. C) The mean change from baseline was significantly higher in stressed rats treated with the LNAmir17-5p inhibitor (n = 6) compared with stressed rats treated with the control scramble (n = 8) indicating an exacerbation of visceral sensitivity *P<0.05 two-way ANOVA followed by Bonferroni post test.
Mentions: Stressed rats treated with the scramble negative control probe showed significant increase of VMR to CRD at day 11 compared with baseline at 60 mmHg. These results are consistent with our previous report that chronic WA induces visceral hyperalgesia[13]. Interestingly, rats exposed to WA stress and receiving the treatment with the miRCURY LNA mir17-5p inhibitor probe exhibited exacerbated increase of the VMR to CRD compared with the scramble control (Fig 8).

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.