MicroRNA-133α regulates neurotensin-associated colonic inflammation in colonic epithelial cells and experimental colitis

Abstract

Ulcerative colitis (UC) and Crohn’s Disease (CD) are the two most common forms of Inflammatory Bowel Diseases (IBD) marked by chronic and persistent inflammation. Neurotensin (NT), together with its receptor, NT receptor 1 (NTR1), are important mediators in intestinal inflammation and their expression is upregulated in the intestine of experimental colitis models and UC colonic biopsies. MicroRNAs (miRNAs) are short, non-coding RNA molecules which act as transcription repressors. We have previously shown that NT exposure upregulates miR-133α expression in human colonocytes NCM460 cells overexpressing NTR1 (NCM460-NTR1). Recently, miR-133α was further examined forits role in NT-associated proinflammatory signaling cascades and acute colitis in vivo. Our study shows that NT-induced miR-133α upregulation modulates NF-κB phosphorylation and promotes proinflammatory cytokine production. In addition, intracolonicinjection of antisense-miR-133α before colitis induction improves histological scores and proinflammatory cytokine transcription. More importantly, dysregulation of miR-133α levels and aftiphilin (AFTPH), a newly-identified miR-133α downstream target, is found only in UC patients, but not in patients with CD. Taken together, we identified NTR1/miR-133α/aftiphilin as a novel regulatory axis involved in NT-associated colonic inflammation in human colonocytes, acute colitis mouse model and in colonic biopsies from UC patients. Our results also provide evidence that colonic levels of NTR1, miR-133α and aftiphilin may also serve as potential biomarkers in UC.

Ulcerative colitis (UC) and Crohn’s disease (CD) are two most common forms of Inflammatory Bowel Diseases (IBD), which are characterized by chronic and persistent inflammation with relapsing and remitting episodes. Neurotensin (NT) is a proinflammatory neuropeptide found in the central nervous system and the intestine ^{[1, 2]}, while NT receptor 1 (NTR1), which has a high affinity with NT, is expressed in neurons ^[3], colonic epithelial ^{[2, 4–6]}, immune cells ^{[7, 8]} and colon cancer cell lines ^[9]. We and others have shown that during intestinal inflammation in experimental colitis mouse models ^{[2, 5, 10, 11]} and in the colon of UC patients ^[5], colonic NT and NTR1 expression is upregulated. Importantly, NT/NTR1 signaling promotes inflammation in colons in animal models induced with acute colitis ^[2,4], while mice deficient in NTR1 have attenuated colitis development and reduced mortality ^{[4, 12]} during the induction of experimental colitis. In addition, NT/NTR1 signaling has been shown to target on ERK and Akt activation in human colonic epithelial cells ^{[6, 9, 13]}. The above evidence suggests that NT/NTR1 signaling is important in colonic inflammation both in colitis models and in human pathophysiology.

MicroRNAs (miRNAs) are short, non-coding RNA molecules which act as transcription repressors. They represent targets of inflammatory agents and act as regulators of genes responsible for immune response ^[14–18]. Several studies demonstrated differential miRNA expression in the circulation ^[19–22] and in the colonic mucosa of UC ^[23–26] and CD ^{[25, 27]} patients as well as in the colon of experimental colitis mouse models ^[24]. However, whether miR-133α was involved in the development and progress of colitis was not known. Recently, we have shown that the binding of NT to NTR1 in human colonocytes NCM460 cells overexpressing NTR1 (NCM460-NTR1) modulates expression levels of several miRNAs^[9], including miR-133α. Interestingly, during colon cancer development, miR-133α and its target genes regulate activation of ERK ^{[28, 29]} and Akt ^{[30, 31]} which are also targeted by NTR1 signaling ^{[6, 9, 13]}.

In our recent study, we investigated how miR-133α was involved in NT-associated colonic inflammation. We first examined miR-133α levels in two human colon epithelial cell lines, the non-transformed NCM460 cells (expressing low NTR1 levels) overexpressing NTR1 (NCM460-NTR1) and the colon cancer HCT116 cells expressing high NTR1 levels. Consistent with our previous findings ^[9], miR-133α expression was upregulated upon NT exposure in both cell lines. We then studied the association of miR-133α expression with colonic inflammation in vivo. NT was administered to wild type C57BL/6 mice through intracolonic administration as a mediator of acute colonic inflammation ^{[2, 12]}. RT-PCR analysis revealed that NT stimulation upregulated miR-133α expression in colon tissues in wild type mice. We also found that miR-133α expression was upregulated in two chemically induced mouse colitis models (2, 4, 6- trinitrobenzenesulfonic acid, TNBS; and dextran sodium sulfate, DSS). Together, these results imply that miR-133α may play a role in intestinal inflammation in vitro and in vivo.

Dysregulation of mucosal homeostasis in IBD is caused by prolonged inflammation in the colonic mucosa. Therefore, we next examined the role of miR-133α in proinflammatory signaling responses in human colonocytes in vitro and in acute experimental colitis in vivo. The differential signaling activated by NTR1 activation in human colonocyte NCM460-NTR1 cells was studied after downregulation of miR-133α in vitro. Our results showed that miR-133α not only regulated ERK activation as previously described ^[28,29], but miR-133α silencing also reduced NF-κB activation and proinflammatory cytokine production. Several studies have shown that localized delivery of antisense oligonucleotides against NF-κB ^[32], TNF alpha ^[33] or miR-141 ^[34] are effective in ameliorating experimental colitis. To examine the impact of miR-133α in vivo we reduced local colonic miR-133α levels by intracolonic injection of antisense-miR-133α oligonucleotides prior to acute colitis induction by TNBS administration. Mice administered with antisense-miR-133α oligonucleotides showed improved histological score and reduced proinflammatory cytokine production. In addition, miR-133α level was mainly upregulated in colonic epithelial cells in mice with colitis and majority of antisense oligonucleotides delivered intracolonically was confined to colonic epithelial cells in vivo ^[34]. Taken together, our data showed that reducing miR-133α expression in colonic epithelial cells through local delivery of antisense- miR-133α oligonucleotides in vivo attenuates development of acute colitis.

As described above miRs inhibit transcription of different genes serving as downstream targets thereby affecting a wide range of diseases, including inflammation ^[35]. MiRs exert their effect by binding 3’untranslated regions (UTRs) of transcript ^[36], thereby repressing deadenylation during translation and promoting mRNA degradation ^[37]. Several downstream targets have been identified for miR-133α that play a role in inflammation (UCP2 ^[38]), cancer (EGFR ^[39,40], GSTP1 ^[41], PNP ^[42], TAGLN2 ^{[42, 43]}) and oxidative stress (GM-CSF ^[44]). Using in silico search in 3 online databases [miRBase (www.mirbase.org) and PicTar (http://pictar.mdc-berlin.de);TargetScanHuman (www.targetscan.org)], we have identified a novel miR-133α target, aftiphilin (AFTPH). NT stimulation of human colonocytes or miR-133α overexpression in these cells lowered AFTPH mRNA levels. The role of AFTPH in cellular function has been studied in a small number of studies. AFTPH contains binding motifs for the structural protein clathrin ^[45] and is localized in trans-golgi network (TGN) ^[46]. Although TGN morphology is not affected by AFTPH gene silencing ^[47], downregulated AFTPH levels promote dysregulated exocytosis of Weibel-Palade bodies in endothelial cells ^[48].

The clinical relevance of our findings was also examined by measuring the levels of miR-133α and its novel downstream target, AFTPH, in colonic cDNA samples from UC and CD patients and their normal controls. We showed that increased miR-133α, but reduced AFTPH transcription were detected in mucosal biopsies of UC, but not CD patients, when compared to normal controls. In addition, we have also investigated the role of AFTPH, a protein responsible for intracellular trafficking ^[45] and exocytosis ^[48], in proinflammatory signaling cascades. Consistent with the results from our miR-133α studies, AFTPH gene-silencing in vitro enhanced NF-κB phosphorylation and IL-1β production in human colonocytes. Therefore, dysregulation in miR-133α and AFTPH expression modulates proinflammatory responses in human colonocytes and possibly in the mucosa in colons during colitis and IBD.

One of the important strategies of drug development in IBD is to limit chronic inflammation. Understanding the mechanism(s) of IBD-related inflammation enables us to target new genes and /or signaling pathways. Our recent study identified a novel NTR1/miR-133α/AFTPH network involved in proinflammatory signaling regulation in colonic epithelial cells in vitro and in vivo, present evidence implying their potential use as biomarkers for UC. Consequently, future work will explore the effect of reducing AFTPH expression in acute colitis development by intracolonic administration of siRNA against AFTPH. In addition, although MyoD and myogenin are the two known transcription factors promoting miR-133α transcription during muscle development ^{[49, 50]}, the molecular mechanisms for regulating miR-133α during inflammation has not been examined. Studies on the mechanism(s) related to the regulation of expression of miR-133α will provide more insights to the proinflammatory response during colitis.