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miR-19b downregulates intestinal SOCS3 to reduce intestinal inflammation in Crohn's disease.

Cheng X, Zhang X, Su J, Zhang Y, Zhou W, Zhou J, Wang C, Liang H, Chen X, Shi R, Zen K, Zhang CY, Zhang H - Sci Rep (2015)

Bottom Line: Furthermore, overexpression of miR-19b decreased SOCS3 expression, leading to increased production of macrophage-inflammatory protein-3α (MIP-3α) in Caco2 cells.In contrast, knockdown of miR-19b increased SOCS3 and decreased MIP-3α.Finally, intracolonically delivered miR-19b decreased the severity of colitis induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China.

ABSTRACT
Although aberrant microRNA (miRNA) expression has frequently been observed in inflammatory bowel disease (IBD), its biological functions and targets remain largely unknown. Present study found that miR-19b was significantly downregulated in active Crohn's disease (CD). Using bioinformatics analysis, suppressor of cytokine signalling 3 (SOCS3), a physiological regulator of innate and adaptive immunity that controls several immuno-inflammatory diseases, was predicted to be a potential target of miR-19b. An inverse correlation between miR-19b and SOCS3 protein levels, but not mRNA, was identified in active-CD intestinal tissue samples. By overexpressing or knocking down miR-19b in Caco2 cells and HT29 cells, it was experimentally validated that miR-19b is a direct regulator of SOCS3. Using a luciferase reporter assay, it was confirmed that miR-19b directly recognizes the 3'-untranslated region (3'-UTR) of SOCS3. Furthermore, overexpression of miR-19b decreased SOCS3 expression, leading to increased production of macrophage-inflammatory protein-3α (MIP-3α) in Caco2 cells. In contrast, knockdown of miR-19b increased SOCS3 and decreased MIP-3α. Finally, intracolonically delivered miR-19b decreased the severity of colitis induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS). Taken together, our findings suggest that miR-19b suppresses the inflammatory response by inhibiting SOCS3 to modulate chemokine production in intestinal epithelial cells (IECs) and thereby prevents the pathogenesis of CD.

No MeSH data available.


Related in: MedlinePlus

SOCS3 protein, but not SOCS3 mRNA, is upregulated in active CD intestinal mucosa tissue. (a), Intestinal SOCS3 expression in normal control and CD patients was determined by immunohistochemical staining. No positive expression was observed in sites implanted with isotype-matched control Ab. The average IOD was obtained by analysing SOCS3 IHC in five random fields of each slide. Left panel: representative image; right panel: quantitative analysis. Results are presented as the mean ± SEM (low magnification, 200×, scale bars = 50 μm; high magnification, 400×, scale bars = 30 μm; n = 20 per group;**p = 0.001104). (b), Western blot analysis of SOCS3 expression in the inflamed jejunum, ileum and colonic mucosa of CD patients and the colon of normal control patients. SOCS3 densitometry quantification. Relative expression was normalized to α-tubulin. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole-blot pictures are available in Supplementary Fig. S5. Results are presented as the mean±SEM (n = 20 per group; ***p = 0.0000, normal colon vs. CD inflamed colon; p = 0.277569, CD inflamed colon vs. CD inflamed ileum; p = 0.158925, CD inflamed colon vs. CD inflamed jejunum). (c), Relative quantification of SOCS3 mRNA normalized to GAPDH in intestinal tissues from control and CD samples (n = 6; p = 0.107).
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f1: SOCS3 protein, but not SOCS3 mRNA, is upregulated in active CD intestinal mucosa tissue. (a), Intestinal SOCS3 expression in normal control and CD patients was determined by immunohistochemical staining. No positive expression was observed in sites implanted with isotype-matched control Ab. The average IOD was obtained by analysing SOCS3 IHC in five random fields of each slide. Left panel: representative image; right panel: quantitative analysis. Results are presented as the mean ± SEM (low magnification, 200×, scale bars = 50 μm; high magnification, 400×, scale bars = 30 μm; n = 20 per group;**p = 0.001104). (b), Western blot analysis of SOCS3 expression in the inflamed jejunum, ileum and colonic mucosa of CD patients and the colon of normal control patients. SOCS3 densitometry quantification. Relative expression was normalized to α-tubulin. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole-blot pictures are available in Supplementary Fig. S5. Results are presented as the mean±SEM (n = 20 per group; ***p = 0.0000, normal colon vs. CD inflamed colon; p = 0.277569, CD inflamed colon vs. CD inflamed ileum; p = 0.158925, CD inflamed colon vs. CD inflamed jejunum). (c), Relative quantification of SOCS3 mRNA normalized to GAPDH in intestinal tissues from control and CD samples (n = 6; p = 0.107).

Mentions: To evaluate SOCS3 expression and distribution in the intestinal mucosa, we performed quantitative RT-PCR, immunohistochemical (IHC) analysis and Western blot analysis. IHC analysis revealed that SOCS3 protein was highly expressed in the intestinal mucosa of active CD patients compared to normal controls, especially in the epithelial layer (Fig. 1a). In addition, the intensity of SOCS3 expression was significantly higher in sites with immobilized SOCS3 antibody, whereas expression was undetectable in sites labelled with isotype-matched control monoclonal antibody (mAb) (Fig. 1a). Based on Western blot analysis, SOCS3 protein was intensely expressed in the inflamed jejunum, ileum and colon mucosa of CD patients, whereas this expression was barely detectable in normal colon mucosa; there was no difference in SOCS3 expression between the jejunum, ileum and colon in CD patients (Fig. 1b). Interestingly, SOCS3 mRNA expression was not significantly different among inflamed colonic tissue and small intestinal mucosa from CD patients and normal intestinal mucosa from control subjects (Fig. 1c). This disparity between SOCS3 protein and mRNA expression in active CD suggests that SOCS3 expression is post-transcriptionally regulated.


miR-19b downregulates intestinal SOCS3 to reduce intestinal inflammation in Crohn's disease.

Cheng X, Zhang X, Su J, Zhang Y, Zhou W, Zhou J, Wang C, Liang H, Chen X, Shi R, Zen K, Zhang CY, Zhang H - Sci Rep (2015)

SOCS3 protein, but not SOCS3 mRNA, is upregulated in active CD intestinal mucosa tissue. (a), Intestinal SOCS3 expression in normal control and CD patients was determined by immunohistochemical staining. No positive expression was observed in sites implanted with isotype-matched control Ab. The average IOD was obtained by analysing SOCS3 IHC in five random fields of each slide. Left panel: representative image; right panel: quantitative analysis. Results are presented as the mean ± SEM (low magnification, 200×, scale bars = 50 μm; high magnification, 400×, scale bars = 30 μm; n = 20 per group;**p = 0.001104). (b), Western blot analysis of SOCS3 expression in the inflamed jejunum, ileum and colonic mucosa of CD patients and the colon of normal control patients. SOCS3 densitometry quantification. Relative expression was normalized to α-tubulin. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole-blot pictures are available in Supplementary Fig. S5. Results are presented as the mean±SEM (n = 20 per group; ***p = 0.0000, normal colon vs. CD inflamed colon; p = 0.277569, CD inflamed colon vs. CD inflamed ileum; p = 0.158925, CD inflamed colon vs. CD inflamed jejunum). (c), Relative quantification of SOCS3 mRNA normalized to GAPDH in intestinal tissues from control and CD samples (n = 6; p = 0.107).
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f1: SOCS3 protein, but not SOCS3 mRNA, is upregulated in active CD intestinal mucosa tissue. (a), Intestinal SOCS3 expression in normal control and CD patients was determined by immunohistochemical staining. No positive expression was observed in sites implanted with isotype-matched control Ab. The average IOD was obtained by analysing SOCS3 IHC in five random fields of each slide. Left panel: representative image; right panel: quantitative analysis. Results are presented as the mean ± SEM (low magnification, 200×, scale bars = 50 μm; high magnification, 400×, scale bars = 30 μm; n = 20 per group;**p = 0.001104). (b), Western blot analysis of SOCS3 expression in the inflamed jejunum, ileum and colonic mucosa of CD patients and the colon of normal control patients. SOCS3 densitometry quantification. Relative expression was normalized to α-tubulin. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole-blot pictures are available in Supplementary Fig. S5. Results are presented as the mean±SEM (n = 20 per group; ***p = 0.0000, normal colon vs. CD inflamed colon; p = 0.277569, CD inflamed colon vs. CD inflamed ileum; p = 0.158925, CD inflamed colon vs. CD inflamed jejunum). (c), Relative quantification of SOCS3 mRNA normalized to GAPDH in intestinal tissues from control and CD samples (n = 6; p = 0.107).
Mentions: To evaluate SOCS3 expression and distribution in the intestinal mucosa, we performed quantitative RT-PCR, immunohistochemical (IHC) analysis and Western blot analysis. IHC analysis revealed that SOCS3 protein was highly expressed in the intestinal mucosa of active CD patients compared to normal controls, especially in the epithelial layer (Fig. 1a). In addition, the intensity of SOCS3 expression was significantly higher in sites with immobilized SOCS3 antibody, whereas expression was undetectable in sites labelled with isotype-matched control monoclonal antibody (mAb) (Fig. 1a). Based on Western blot analysis, SOCS3 protein was intensely expressed in the inflamed jejunum, ileum and colon mucosa of CD patients, whereas this expression was barely detectable in normal colon mucosa; there was no difference in SOCS3 expression between the jejunum, ileum and colon in CD patients (Fig. 1b). Interestingly, SOCS3 mRNA expression was not significantly different among inflamed colonic tissue and small intestinal mucosa from CD patients and normal intestinal mucosa from control subjects (Fig. 1c). This disparity between SOCS3 protein and mRNA expression in active CD suggests that SOCS3 expression is post-transcriptionally regulated.

Bottom Line: Furthermore, overexpression of miR-19b decreased SOCS3 expression, leading to increased production of macrophage-inflammatory protein-3α (MIP-3α) in Caco2 cells.In contrast, knockdown of miR-19b increased SOCS3 and decreased MIP-3α.Finally, intracolonically delivered miR-19b decreased the severity of colitis induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS).

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China.

ABSTRACT
Although aberrant microRNA (miRNA) expression has frequently been observed in inflammatory bowel disease (IBD), its biological functions and targets remain largely unknown. Present study found that miR-19b was significantly downregulated in active Crohn's disease (CD). Using bioinformatics analysis, suppressor of cytokine signalling 3 (SOCS3), a physiological regulator of innate and adaptive immunity that controls several immuno-inflammatory diseases, was predicted to be a potential target of miR-19b. An inverse correlation between miR-19b and SOCS3 protein levels, but not mRNA, was identified in active-CD intestinal tissue samples. By overexpressing or knocking down miR-19b in Caco2 cells and HT29 cells, it was experimentally validated that miR-19b is a direct regulator of SOCS3. Using a luciferase reporter assay, it was confirmed that miR-19b directly recognizes the 3'-untranslated region (3'-UTR) of SOCS3. Furthermore, overexpression of miR-19b decreased SOCS3 expression, leading to increased production of macrophage-inflammatory protein-3α (MIP-3α) in Caco2 cells. In contrast, knockdown of miR-19b increased SOCS3 and decreased MIP-3α. Finally, intracolonically delivered miR-19b decreased the severity of colitis induced with 2,4,6-trinitrobenzene sulphonic acid (TNBS). Taken together, our findings suggest that miR-19b suppresses the inflammatory response by inhibiting SOCS3 to modulate chemokine production in intestinal epithelial cells (IECs) and thereby prevents the pathogenesis of CD.

No MeSH data available.


Related in: MedlinePlus