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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.


Related in: MedlinePlus

Expression of mi17-5p in the spinal cord assessed by in situ hybridization with co-immunofluorescence staining for astrocytes (GFAP) and DAPI.A) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from control rats. B) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from stressed rats. Arrows indicate evidence of mir-17-5p staining in peri-nuclear space colocalizing with astrocytes. Scale car = 50mM. C) In situ hybridization with scramble negative control probe with co-immunofluorescence staining for astrocytes (GFAP) in the dorsal horn of the spinal cord from a stressed rat. Scale bar = 200 mM.
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pone.0130938.g007: Expression of mi17-5p in the spinal cord assessed by in situ hybridization with co-immunofluorescence staining for astrocytes (GFAP) and DAPI.A) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from control rats. B) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from stressed rats. Arrows indicate evidence of mir-17-5p staining in peri-nuclear space colocalizing with astrocytes. Scale car = 50mM. C) In situ hybridization with scramble negative control probe with co-immunofluorescence staining for astrocytes (GFAP) in the dorsal horn of the spinal cord from a stressed rat. Scale bar = 200 mM.

Mentions: The expression and localization of mir17-5p in the spinal cord was evaluated using in situ hybridization for mir17-5p. Sections stained with the NBT-BCP detection system showed high mir17-5p expression in the dorsal horn of the spinal cord from stressed rats (Fig 6). When using the HNPP Fluorescent detection set for fluorescent detection of mir17-5p combined with immunofluorecent labeling for GFAP for astrocytes, staining for mir17-5p was verified in sections from stressed animals and while the expression was observed throughout the spinal cord, mir17-5p was also clearly expressed in the superficial laminae of the dorsal horn spinal cord where nociceptive fibers from the gut are distributed. The co-immunostaining with GFAP revealed co-localization of mir17-5p with astrocytes, but also in the peri-nuclear region of other cells as shown by mir17-5p staining in DAPI positive cells (Fig 7A and 7B). As expected, sections treated with the scramble negative control probe showed no specific staining (Fig 7C)


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)

Expression of mi17-5p in the spinal cord assessed by in situ hybridization with co-immunofluorescence staining for astrocytes (GFAP) and DAPI.A) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from control rats. B) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from stressed rats. Arrows indicate evidence of mir-17-5p staining in peri-nuclear space colocalizing with astrocytes. Scale car = 50mM. C) In situ hybridization with scramble negative control probe with co-immunofluorescence staining for astrocytes (GFAP) in the dorsal horn of the spinal cord from a stressed rat. Scale bar = 200 mM.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130938.g007: Expression of mi17-5p in the spinal cord assessed by in situ hybridization with co-immunofluorescence staining for astrocytes (GFAP) and DAPI.A) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from control rats. B) In situ hybridization of mir17-5p (red) with co-immunostaining for GFAP (green) and DAPI (blue) in the dorsal horn of the spinal cord from stressed rats. Arrows indicate evidence of mir-17-5p staining in peri-nuclear space colocalizing with astrocytes. Scale car = 50mM. C) In situ hybridization with scramble negative control probe with co-immunofluorescence staining for astrocytes (GFAP) in the dorsal horn of the spinal cord from a stressed rat. Scale bar = 200 mM.
Mentions: The expression and localization of mir17-5p in the spinal cord was evaluated using in situ hybridization for mir17-5p. Sections stained with the NBT-BCP detection system showed high mir17-5p expression in the dorsal horn of the spinal cord from stressed rats (Fig 6). When using the HNPP Fluorescent detection set for fluorescent detection of mir17-5p combined with immunofluorecent labeling for GFAP for astrocytes, staining for mir17-5p was verified in sections from stressed animals and while the expression was observed throughout the spinal cord, mir17-5p was also clearly expressed in the superficial laminae of the dorsal horn spinal cord where nociceptive fibers from the gut are distributed. The co-immunostaining with GFAP revealed co-localization of mir17-5p with astrocytes, but also in the peri-nuclear region of other cells as shown by mir17-5p staining in DAPI positive cells (Fig 7A and 7B). As expected, sections treated with the scramble negative control probe showed no specific staining (Fig 7C)

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.


Related in: MedlinePlus