MicroRNA-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis

Abstract

Substance P (SP) mediates colitis. SP signaling regulates the expression of several miRNAs, including miR-31-3p, in human colonocytes. However, the role of miR-31-3p in colitis and the underlying mechanisms has not been elucidated. We performed real-time PCR analysis of miR-31-3p expression in human colonic epithelial cells overexpressing neurokinin-1 receptor (NCM460 NK-1R) in response to SP stimulation and in NCM460 cells after IL-6, IL8, tumor necrosis factor (TNF)-α, and interferon-γ exposure. Functions of miR-31-3p were tested in NCM460-NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis. Targets of miRNA-31-3p were confirmed by Western blot analysis and luciferase reporter assay. Jun N-terminal kinase inhibition decreased SP-induced miR-31-3p expression. miR-31-3p expression was increased in both TNBS- and DSS-induced colitis and human colonic biopsies from ulcerative colitis, compared with controls. Intracolonic administration of a miR-31-3p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic TNF-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) mRNA expression. Conversely, overexpression of miR-31-3p ameliorated the severity of DSS-induced colitis. Bioinformatic, luciferase reporter assay, and Western blot analyses identified RhoA as a target of miR-31-3p in NCM460 cells. Constitutive activation of RhoA led to increased expression of CCL2, IL6, TNF-α, and CXCL10 in NCM460-NK-1R cells on SP stimulation. Our results reveal a novel SP-miR-31-3p-RhoA pathway that protects from colitis. The use of miR-31-3p mimics may be a promising approach for colitis treatment.

Figure 1

Substance P (SP)-regulated miR-31-3p expression in colon epithelial cells. A: SP stimulates miR-31-3p expression in NCM460–neurokinin-1 receptor (NK-1R) cells. B–F: miR-31-3p is up-regulated by IL-6 (B), interferon (IFN)-γ (C), IL-8 (D), tumor necrosis factor (TNF)-α (E), and cytokine cocktail (F) in NCM460 cells. n = 3. ^∗P < 0.05 versus no SP or cytokine-stimulated cells.

Figure 2

Substance P (SP) regulates miR-31-3p expression through a Jun amino-terminal kinase (JNK) signaling pathway. A: Expression levels of miR-31-3p in SP-stimulated NCM460–neurokinin-1 receptor (NK-1R) cells pretreated with the JNK inhibitor (JNKI) SP600125. B: Expression levels of c-Jun and β-actin protein are assessed by Western blot analysis after NCM460–NK-1R cells were transfected with siRNA–c-Jun or control siRNA for 48 hours. C: Expression levels of miR-31-3p in SP-stimulated NCM460–NK-1R cells transfected with siRNA–c-Jun for 48 hours before SP exposure for 6 hours. Data are expressed as means ± SD. n = 3 samples per experimental condition. ^∗P < 0.05.

Figure 3

miR-31-3p regulates inflammatory gene expression in colonic epithelial cells. A and B: miR-31-3p down-regulation increases chemokine (C-C motif) ligand 2 (CCL2) (A) and IL-6 (B) mRNA expression in response to substance P (SP) stimulation. C and D: miR-31-3p overexpression decreases CCL2 (C) and IL-6 (D) mRNA expression in response to SP stimulation. E and F: miR-31-3p overexpression decreases CCL2 (E) and IL-6 (F) mRNA expression after incubation with a cytokine cocktail. G and H: miR-31-3p overexpression decreases IL-1B (G) and tumor necrosis factor (TNF)-α (H) mRNA expression in response to a cytokine cocktail. Data are expressed as means ± SD. n = 3.^∗P < 0.05. As, antisense; con, control.

Figure 4

miR-31-3p expression increases in ulcerative colitis (UC) tissues and experimental colitis. A: Expression of miR-31-3p in human UC tissue samples compared with control tissue samples. B: Representative images of in situ hybridization of miR-31-3p of colon tissues from UC patients and controls (arrow indicates epithelial cells). C: Expression of miR-31-3p in human Crohn disease (CD) tissue samples compared with control tissue samples is shown. D: Representative images of in situ hybridization of miR-31-3p of colon tissues from CD patients and controls (arrow indicates epithelial cells). E: Expression of miR-31-3p increases in dextran sodium sulfate (DSS) colitis. F: Representative images of in situ hybridization of miR-31-3p of colon tissues from DSS-treated C57BL6/J mice and their control counterparts (arrow indicates epithelial cells). G: Expression of miR-31-3p in colon tissues from neurokinin-1 receptor (NK-1R) knockout (KO) mice and their control counterparts [wild-type (WT) mice] in trinitrobenzene sulfonic acid (TNBS)-induced colitis. H: Representative images of in situ hybridization of miR-31-3p of colon tissues from wild-type mice with or without TNBS treatment (arrow indicates epithelial cells). Data are expressed as means ± SD. n = 14 human UC tissue samples (A); n = 10 control tissue samples (A); n = 15 human CD tissue samples (C); n = 9 control tissue samples (C); n = 8 control tissue samples (E); n = 7 DSS colitis samples (E); n = 5 (G). P > 0.05 control versus CD (C). ^∗P < 0.05 versus control. Scale bars = 50 μm.

Figure 5

Antisense (as)-miR-31-3p exacerbates trinitrobenzene sulfonic acid (TNBS)-induced colitis. A: Timeline of as-miR-31-3p treatment in TNBS-induced colitis. B: Percentage of initial body weight of each group of mice during treatment. C: Expression level of miR-31-3p was assessed by real-time PCR after intracolonic administration of as-miR-31-3p. D: Histopathology score of as-miR-31-3p–treated mice with TNBS-induced colitis. E: TNBS-induced histologic changes in colons of controls or as-miR-31-3p–treated C57BL6/J mice. F–H: Colon length, colon weight, and colon weight-to-length ratio of each group of mice. I: Expression of tumor necrosis factor (TNF)-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) in the colon tissue of as-miR-31-3p– or as-miR-control–treated TNBS mice colitis models. Data are expressed as means ± SD. n = 8. ^∗P < 0.05. Scale bar = 100 μm. Con, control.

Figure 6

Antisense (as)-miR-31-3p exacerbates dextran sodium sulfate (DSS)-induced colitis. A: Timeline of as-miR-31-3p treatment in 2% DSS-induced colitis. B: Percentage of initial body weight of each group of mice during treatment. C: Histologic changes in colons of controls or as-miR-31-3p–treated C57BL6/J mice in DSS colitis model. D: Expression level of miR-31-3p after intracolonic administration of as-miR-31-3p. E: Colon length of each group of mice. F: Disease activity index scores of as-miR-31-3p–treated mice with DSS-induced colitis. ^∗G: Histopathology score of as-miR-31-3p–treated mice with DSS-induced colitis. H: Crypt damage score of as-miR-31-3p–treated mice with DSS-induced colitis. I: Edema score of as-miR-31-3p–treated mice with DSS-induced colitis. J: Expression of tumor necrosis factor (TNF)-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) in the colon tissue of as-miR-31-3p– or anti-miR-control–treated DSS-induced colitis models. Data are expressed as means ± SD. n = 8. ^∗P < 0.05. Scale bar = 100 μm.

Figure 7

miR-31-3p mimic treatment reduces the intestinal inflammatory response in dextran sodium sulfate (DSS)-induced colitis. A: Timeline of miR-31-3p mimic treatment in 2% DSS-induced colitis. B: Percentage of initial body weight of each group of mice during treatment. C: Histologic changes in colons of controls or miR-31-3p mimic–treated C57BL6/J mice in DSS colitis model. D: Expression level of miR-31-3p after intracolonic administration of miR-31-3p mimic. E: Colon length of each group of mice. F: Colon weight of each group of mice. G: Disease activity index scores of miR-31-3p mimic–treated mice with DSS-induced colitis. H: Histopathology score of miR-31-3p mimic–treated mice with DSS-induced colitis. I: Polymorphonuclear leukocyte (PMN) infiltration score of miR-31-3p mimic–treated mice with DSS-induced colitis. J: Expression of tumor necrosis factor (TNF)-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) in the colon tissue of miR-31-3p mimic– or control mimic–treated DSS-induced colitis models. Data are expressed as means ± SD. n = 8. ^∗P < 0.05. Scale bar = 100 μm.

Figure 8

miR-31-3p targets RhoA in colonic epithelial cells. A: Human RhoA mRNA contains a predicted miR-31-3p binding site. B: Overexpression of miR-31-3p inhibits RhoA protein expression in NCM460–neurokinin-1 receptor (NK-1R) cells. C: Overexpression of miR-31-3p inhibits RhoA protein activation in response to substance P (SP) stimulation for 10 minutes in NCM460–NK-1R cells. D: Wild-type (WT) or mutant (Mut) RhoA 3′ untranslated region (UTR) luciferase constructs were cotransfected with miR-31-3p mimic or control mimic in NCM460–NK-1R cells. Luciferase activity was determined 24 hours after transfection, and normalized luciferase activity is shown. E: Chemokine (C-C motif) ligand 2 (CCL2), IL-6, tumor necrosis factor (TNF)-α, and CXCL10 mRNA expression in response to SP stimulation of NCM460–NK-1R cells with constitutive activated RhoA. Data are expressed as means ± SD. n = 3 experiments. ^∗P < 0.05 versus control; ^†P < 0.05. Ad, adenovirus.

Figure 9

Schematic representation of proposed substance P (SP)-miR-31-3p signaling pathway in human colonic epithelial cells. After binding to the neurokinin-1 receptor (NK-1R), SP stimulates miR-31-3p by c-Jun N-terminal kinase (JNK) activation in human colonic epithelial cells. miR-31-3p regulates inflammation by down-regulating RhoA expression.