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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

The relationship between miR-19b and SOCS3 in the pathogenesis of colitis(a), Quantitative real-time PCR analysis of miR-19b levels in different treated groups. (n = 8 mice per group;*p = 0.021030, control group vs. TNBS group;***p = 0.000628, TNBS group vs. PEI+pre-miR19b group; *p = 0.022823, PEI group vs. PEI+pre-miR-19b group;***p = 0.000837, PEI+pre-scramble group vs. PEI+pre-miR-19b group). (b), The Western blot analysis of colon sections for different treated mice implanted with SOCS3 antibody is presented. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole blot pictures are available in Supplementary Fig. S7. (n = 8 mice per group;***p = 0.0000, normal control vs. PEI+pre-miR19b group;***p = 0.000015, TNBS group vs. PEI+pre-miR19b group;***p = 0.000010, PEI+pre-scramble group vs. PEI+pre-miR-19b group; ***p = 0.0000, PEI group vs. PEI+pre-miR-19b group). (c), SOCS3 mRNA was normalized to GAPDH expression in colon tissues of treated mice by PCR. None significance were detected in all groups. (d), Tissue homogenates from each group mice were subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein (n = 8 mice per group;**p = 0.002473, control group vs. TNBS group;***p = 0.000077, TNBS group vs. PEI+pre-miR19b group;***p = 0.000015, PEI group vs. PEI+pre-miR-19b group;***p = 0.00013, PEI+pre-scramble group vs. PEI+pre-miR-19b group).
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f7: The relationship between miR-19b and SOCS3 in the pathogenesis of colitis(a), Quantitative real-time PCR analysis of miR-19b levels in different treated groups. (n = 8 mice per group;*p = 0.021030, control group vs. TNBS group;***p = 0.000628, TNBS group vs. PEI+pre-miR19b group; *p = 0.022823, PEI group vs. PEI+pre-miR-19b group;***p = 0.000837, PEI+pre-scramble group vs. PEI+pre-miR-19b group). (b), The Western blot analysis of colon sections for different treated mice implanted with SOCS3 antibody is presented. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole blot pictures are available in Supplementary Fig. S7. (n = 8 mice per group;***p = 0.0000, normal control vs. PEI+pre-miR19b group;***p = 0.000015, TNBS group vs. PEI+pre-miR19b group;***p = 0.000010, PEI+pre-scramble group vs. PEI+pre-miR-19b group; ***p = 0.0000, PEI group vs. PEI+pre-miR-19b group). (c), SOCS3 mRNA was normalized to GAPDH expression in colon tissues of treated mice by PCR. None significance were detected in all groups. (d), Tissue homogenates from each group mice were subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein (n = 8 mice per group;**p = 0.002473, control group vs. TNBS group;***p = 0.000077, TNBS group vs. PEI+pre-miR19b group;***p = 0.000015, PEI group vs. PEI+pre-miR-19b group;***p = 0.00013, PEI+pre-scramble group vs. PEI+pre-miR-19b group).

Mentions: To investigate the relationship between miR-19b and SOCS3 in the pathogenesis of colitis, we employed the TNBS-induced colitis model and examined SOCS3 expression after intra-colonic pre-miR-19b, pre-scramble or 50% ethanol administration. As shown in Fig. 7a,b, the expression of miR-19b in TNBS-induced colitis was decreased, while the expression of SOCS3 in TNBS-induced colitis was increased compared with the control. However, SOCS3 protein in TNBS-induced colitis was decreased after intra-colonic administration with pre-miR-19b. When we analysed the alteration in SOCS3 mRNA before and after administration of pre-miR-19b, there was no significant alteration in SOCS3 mRNA after treatment with pre-miR-19b (Fig. 7c). Furthermore, the expression of MIP-3α in TNBS-induced colitis was increased after intra-colonic administration with pre-miR-19b (Fig. 7d). Together, these results suggest that miR-19b regulates SOCS3 protein expression and MIP-3α expression in TNBS-induced colitis.


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)

The relationship between miR-19b and SOCS3 in the pathogenesis of colitis(a), Quantitative real-time PCR analysis of miR-19b levels in different treated groups. (n = 8 mice per group;*p = 0.021030, control group vs. TNBS group;***p = 0.000628, TNBS group vs. PEI+pre-miR19b group; *p = 0.022823, PEI group vs. PEI+pre-miR-19b group;***p = 0.000837, PEI+pre-scramble group vs. PEI+pre-miR-19b group). (b), The Western blot analysis of colon sections for different treated mice implanted with SOCS3 antibody is presented. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole blot pictures are available in Supplementary Fig. S7. (n = 8 mice per group;***p = 0.0000, normal control vs. PEI+pre-miR19b group;***p = 0.000015, TNBS group vs. PEI+pre-miR19b group;***p = 0.000010, PEI+pre-scramble group vs. PEI+pre-miR-19b group; ***p = 0.0000, PEI group vs. PEI+pre-miR-19b group). (c), SOCS3 mRNA was normalized to GAPDH expression in colon tissues of treated mice by PCR. None significance were detected in all groups. (d), Tissue homogenates from each group mice were subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein (n = 8 mice per group;**p = 0.002473, control group vs. TNBS group;***p = 0.000077, TNBS group vs. PEI+pre-miR19b group;***p = 0.000015, PEI group vs. PEI+pre-miR-19b group;***p = 0.00013, PEI+pre-scramble group vs. PEI+pre-miR-19b group).
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f7: The relationship between miR-19b and SOCS3 in the pathogenesis of colitis(a), Quantitative real-time PCR analysis of miR-19b levels in different treated groups. (n = 8 mice per group;*p = 0.021030, control group vs. TNBS group;***p = 0.000628, TNBS group vs. PEI+pre-miR19b group; *p = 0.022823, PEI group vs. PEI+pre-miR-19b group;***p = 0.000837, PEI+pre-scramble group vs. PEI+pre-miR-19b group). (b), The Western blot analysis of colon sections for different treated mice implanted with SOCS3 antibody is presented. Upper panel: representative image; lower panel: quantitative analysis from three independent experiments. The original whole blot pictures are available in Supplementary Fig. S7. (n = 8 mice per group;***p = 0.0000, normal control vs. PEI+pre-miR19b group;***p = 0.000015, TNBS group vs. PEI+pre-miR19b group;***p = 0.000010, PEI+pre-scramble group vs. PEI+pre-miR-19b group; ***p = 0.0000, PEI group vs. PEI+pre-miR-19b group). (c), SOCS3 mRNA was normalized to GAPDH expression in colon tissues of treated mice by PCR. None significance were detected in all groups. (d), Tissue homogenates from each group mice were subjected to ELISA to quantify MIP-3α protein levels. Results are expressed as ng of MIP-3α per mg of total homogenate protein (n = 8 mice per group;**p = 0.002473, control group vs. TNBS group;***p = 0.000077, TNBS group vs. PEI+pre-miR19b group;***p = 0.000015, PEI group vs. PEI+pre-miR-19b group;***p = 0.00013, PEI+pre-scramble group vs. PEI+pre-miR-19b group).
Mentions: To investigate the relationship between miR-19b and SOCS3 in the pathogenesis of colitis, we employed the TNBS-induced colitis model and examined SOCS3 expression after intra-colonic pre-miR-19b, pre-scramble or 50% ethanol administration. As shown in Fig. 7a,b, the expression of miR-19b in TNBS-induced colitis was decreased, while the expression of SOCS3 in TNBS-induced colitis was increased compared with the control. However, SOCS3 protein in TNBS-induced colitis was decreased after intra-colonic administration with pre-miR-19b. When we analysed the alteration in SOCS3 mRNA before and after administration of pre-miR-19b, there was no significant alteration in SOCS3 mRNA after treatment with pre-miR-19b (Fig. 7c). Furthermore, the expression of MIP-3α in TNBS-induced colitis was increased after intra-colonic administration with pre-miR-19b (Fig. 7d). Together, these results suggest that miR-19b regulates SOCS3 protein expression and MIP-3α expression in TNBS-induced colitis.

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