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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

Validation of human SOCS3 as a direct miR-19b target.(a), Quantitative real-time PCR analysis of miR-19b expression in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. Data are presented as the mean±SEM from three independent experiments (Caco2 group: ***p = 0.000092, ncRNA vs. pre-miR-19b, ***p = 0.000031, ncRNA vs. anti-miR-19b; HT29 group: ***p = 0.000253, ncRNA vs. pre-miR-19b,**p = 0.003, ncRNA vs. anti-miR-19b). (b), Immunoblot for endogenous SOCS3 in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. α-Tubulin was used as a loading control. Top panel: representative image; Bottom panel: quantitative analysis. The original whole-blot pictures are available in Supplementary Fig. S6. Results are presented as the mean±SEM from three independent experiments (Caco2 group: **p = 0.0014, ncRNA vs. pre-miR-19b,***p = 0.00036,ncRNA vs. anti-miR-19b; HT29 group: *p = 0.038, ncRNA vs. pre-miR-19b,**p = 0.007, ncRNA vs. anti-miR-19b). (c), Quantitative real-time PCR analysis of SOCS3 expression in Caco2 cells and HT29 cells treated with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. The y-axis shows relative SOCS3 mRNA levels normalized to GAPDH. The results are presented as the mean±SEM from three independent experiments (p > 0.05 vs. ncRNA). (d), Direct recognition of the SOCS3 3’-UTR by miR-19b. Firefly luciferase reporters containing wild-type (WT) or mutant (MT) miR-19b binding sites in the SOCS3 3’-UTR were co-transfected into Caco2 cells and HT29 cells with scrambled ncRNA, pre-miR-19b or anti-miR-19b. Twenty-four hours after transfection, a luciferase assay was performed. Firefly luciferase values were normalized to β-galactosidase activity and plotted as relative luciferase activity. The results were calculated as the ratio of firefly luciferase activity in miR-19b-transfected cells to the luciferase activity in control cells. Left: Caco2 cells. Right: HT29 cells. The results are presented as the mean±SEM from three independent experiments (Caco2 group: *p = 0.013, pre-scramble vs. pre-miR-19b,***p = 0.0000, anti-scramble vs. anti-miR-19b; HT29 group:***p = 0.000021, pre-scramble vs. pre-miR-19b,***p = 0.000019, anti-scramble vs. anti-miR-19b).
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f4: Validation of human SOCS3 as a direct miR-19b target.(a), Quantitative real-time PCR analysis of miR-19b expression in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. Data are presented as the mean±SEM from three independent experiments (Caco2 group: ***p = 0.000092, ncRNA vs. pre-miR-19b, ***p = 0.000031, ncRNA vs. anti-miR-19b; HT29 group: ***p = 0.000253, ncRNA vs. pre-miR-19b,**p = 0.003, ncRNA vs. anti-miR-19b). (b), Immunoblot for endogenous SOCS3 in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. α-Tubulin was used as a loading control. Top panel: representative image; Bottom panel: quantitative analysis. The original whole-blot pictures are available in Supplementary Fig. S6. Results are presented as the mean±SEM from three independent experiments (Caco2 group: **p = 0.0014, ncRNA vs. pre-miR-19b,***p = 0.00036,ncRNA vs. anti-miR-19b; HT29 group: *p = 0.038, ncRNA vs. pre-miR-19b,**p = 0.007, ncRNA vs. anti-miR-19b). (c), Quantitative real-time PCR analysis of SOCS3 expression in Caco2 cells and HT29 cells treated with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. The y-axis shows relative SOCS3 mRNA levels normalized to GAPDH. The results are presented as the mean±SEM from three independent experiments (p > 0.05 vs. ncRNA). (d), Direct recognition of the SOCS3 3’-UTR by miR-19b. Firefly luciferase reporters containing wild-type (WT) or mutant (MT) miR-19b binding sites in the SOCS3 3’-UTR were co-transfected into Caco2 cells and HT29 cells with scrambled ncRNA, pre-miR-19b or anti-miR-19b. Twenty-four hours after transfection, a luciferase assay was performed. Firefly luciferase values were normalized to β-galactosidase activity and plotted as relative luciferase activity. The results were calculated as the ratio of firefly luciferase activity in miR-19b-transfected cells to the luciferase activity in control cells. Left: Caco2 cells. Right: HT29 cells. The results are presented as the mean±SEM from three independent experiments (Caco2 group: *p = 0.013, pre-scramble vs. pre-miR-19b,***p = 0.0000, anti-scramble vs. anti-miR-19b; HT29 group:***p = 0.000021, pre-scramble vs. pre-miR-19b,***p = 0.000019, anti-scramble vs. anti-miR-19b).

Mentions: We further examined the correlation between miR-19b and SOCS3 by altering the miR-19b level and examining the effect on SOCS3 expression in Caco2 cells and HT29 cells. We first overexpressed miR-19b by transfecting cells with pre-miR-19b, a synthetic RNA oligonucleotide duplex mimicking the miR-19b precursor. Then, we knocked down miR-19b by transfecting cells with anti-miR-19b, a chemically modified single-stranded antisense oligonucleotide designed to specifically target mature miR-19b. We validated these changes in miR-9b expression by quantitative RT-PCR 24 h after transfection (Fig. 4a).


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)

Validation of human SOCS3 as a direct miR-19b target.(a), Quantitative real-time PCR analysis of miR-19b expression in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. Data are presented as the mean±SEM from three independent experiments (Caco2 group: ***p = 0.000092, ncRNA vs. pre-miR-19b, ***p = 0.000031, ncRNA vs. anti-miR-19b; HT29 group: ***p = 0.000253, ncRNA vs. pre-miR-19b,**p = 0.003, ncRNA vs. anti-miR-19b). (b), Immunoblot for endogenous SOCS3 in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. α-Tubulin was used as a loading control. Top panel: representative image; Bottom panel: quantitative analysis. The original whole-blot pictures are available in Supplementary Fig. S6. Results are presented as the mean±SEM from three independent experiments (Caco2 group: **p = 0.0014, ncRNA vs. pre-miR-19b,***p = 0.00036,ncRNA vs. anti-miR-19b; HT29 group: *p = 0.038, ncRNA vs. pre-miR-19b,**p = 0.007, ncRNA vs. anti-miR-19b). (c), Quantitative real-time PCR analysis of SOCS3 expression in Caco2 cells and HT29 cells treated with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. The y-axis shows relative SOCS3 mRNA levels normalized to GAPDH. The results are presented as the mean±SEM from three independent experiments (p > 0.05 vs. ncRNA). (d), Direct recognition of the SOCS3 3’-UTR by miR-19b. Firefly luciferase reporters containing wild-type (WT) or mutant (MT) miR-19b binding sites in the SOCS3 3’-UTR were co-transfected into Caco2 cells and HT29 cells with scrambled ncRNA, pre-miR-19b or anti-miR-19b. Twenty-four hours after transfection, a luciferase assay was performed. Firefly luciferase values were normalized to β-galactosidase activity and plotted as relative luciferase activity. The results were calculated as the ratio of firefly luciferase activity in miR-19b-transfected cells to the luciferase activity in control cells. Left: Caco2 cells. Right: HT29 cells. The results are presented as the mean±SEM from three independent experiments (Caco2 group: *p = 0.013, pre-scramble vs. pre-miR-19b,***p = 0.0000, anti-scramble vs. anti-miR-19b; HT29 group:***p = 0.000021, pre-scramble vs. pre-miR-19b,***p = 0.000019, anti-scramble vs. anti-miR-19b).
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f4: Validation of human SOCS3 as a direct miR-19b target.(a), Quantitative real-time PCR analysis of miR-19b expression in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. Data are presented as the mean±SEM from three independent experiments (Caco2 group: ***p = 0.000092, ncRNA vs. pre-miR-19b, ***p = 0.000031, ncRNA vs. anti-miR-19b; HT29 group: ***p = 0.000253, ncRNA vs. pre-miR-19b,**p = 0.003, ncRNA vs. anti-miR-19b). (b), Immunoblot for endogenous SOCS3 in Caco2 cells and HT29 cells transfected with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. α-Tubulin was used as a loading control. Top panel: representative image; Bottom panel: quantitative analysis. The original whole-blot pictures are available in Supplementary Fig. S6. Results are presented as the mean±SEM from three independent experiments (Caco2 group: **p = 0.0014, ncRNA vs. pre-miR-19b,***p = 0.00036,ncRNA vs. anti-miR-19b; HT29 group: *p = 0.038, ncRNA vs. pre-miR-19b,**p = 0.007, ncRNA vs. anti-miR-19b). (c), Quantitative real-time PCR analysis of SOCS3 expression in Caco2 cells and HT29 cells treated with pre-miR-19b, anti-miR-19b or ncRNA. Left: Caco2 cells. Right: HT29 cells. The y-axis shows relative SOCS3 mRNA levels normalized to GAPDH. The results are presented as the mean±SEM from three independent experiments (p > 0.05 vs. ncRNA). (d), Direct recognition of the SOCS3 3’-UTR by miR-19b. Firefly luciferase reporters containing wild-type (WT) or mutant (MT) miR-19b binding sites in the SOCS3 3’-UTR were co-transfected into Caco2 cells and HT29 cells with scrambled ncRNA, pre-miR-19b or anti-miR-19b. Twenty-four hours after transfection, a luciferase assay was performed. Firefly luciferase values were normalized to β-galactosidase activity and plotted as relative luciferase activity. The results were calculated as the ratio of firefly luciferase activity in miR-19b-transfected cells to the luciferase activity in control cells. Left: Caco2 cells. Right: HT29 cells. The results are presented as the mean±SEM from three independent experiments (Caco2 group: *p = 0.013, pre-scramble vs. pre-miR-19b,***p = 0.0000, anti-scramble vs. anti-miR-19b; HT29 group:***p = 0.000021, pre-scramble vs. pre-miR-19b,***p = 0.000019, anti-scramble vs. anti-miR-19b).
Mentions: We further examined the correlation between miR-19b and SOCS3 by altering the miR-19b level and examining the effect on SOCS3 expression in Caco2 cells and HT29 cells. We first overexpressed miR-19b by transfecting cells with pre-miR-19b, a synthetic RNA oligonucleotide duplex mimicking the miR-19b precursor. Then, we knocked down miR-19b by transfecting cells with anti-miR-19b, a chemically modified single-stranded antisense oligonucleotide designed to specifically target mature miR-19b. We validated these changes in miR-9b expression by quantitative RT-PCR 24 h after transfection (Fig. 4a).

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