Oncomir MicroRNA-346 Is Upregulated in Colons of Patients With Primary Sclerosing Cholangitis

Abstract

Introduction: Primary sclerosing cholangitis (PSC) is a cholestatic liver disorder that is frequently associated with ulcerative colitis (UC). Patients with PSC and UC (PSC-UC) have a higher risk of colorectal neoplasia compared with patients with UC. The oncogenic properties of microRNA-346 (miR-346) have been recently reported. We investigated the expression of miR-346 and its 2 target genes, the receptor of vitamin D (VDR), and the tumor necrosis factor-α (TNF-α), which are known to modulate carcinogenesis.

Methods: Ascending and sigmoid colon biopsies were obtained from patients with PSC, PSC and UC (PSC-UC), UC, and healthy controls (n = 10 in each group). Expressions of VDR, TNF-α, 18S RNA, p27, miR-346, and reference microRNA, miR-191, were evaluated by real-time PCR using human TaqMan Gene Expression and TaqMan MicroRNA Assays. Functional studies with miR-346 mimic and inhibitor were conducted in HepG2 and Caco-2 cells. The effect of ursodeoxycholic acid on miR-346 expression was examined in Caco-2 cells.

Results: An increased expression of miR-346 in the ascending colon of PSC-UC was observed (P < 0.001 vs all groups). In patients with UC, an exceptionally low colonic expression of miRNA-346 was accompanied by the extensive upregulation of VDR and TNF-α genes. A functional in vitro analysis demonstrated that inhibition of miR-346 resulted in the upregulation of VDR and TNF-α, whereas the induction of miR-346 activity suppressed VDR, TNF-α, and p27.

Discussion: The upregulation of miRNA-346 in the colon of patients with PSC may be responsible for the disturbance of VDR and TNF-α signaling pathway, which could result in an inadequate suppression of neoplasia.

Figure 1.

Expression of miRNA-346, TNF-α, VDR, and p27 ^kip1 in the ascending colon. In patients with PSC-UC, the expression of miRNA-346 (a) was increased, whereas the levels of VDR protein (c) and TNF-α protein (d) were not changed in comparison to controls. The expression cell cycle inhibitor p27 ^kip1 (e) was substantially inhibited both in patients with PSC-UC and PSC. In contrast, in patients with UC, the miRNA-346 expression (a) was drastically suppressed, and it was accompanied by the enhanced expression of TNF-α mRNA (b), VDR protein (c), and TNF-α protein (d), whereas the expression cell cycle inhibitor p27 ^kip1 (e) was comparable to control values. MicroRNA-191 served as reference microRNA. Results are representative of n = 10 independent experiments per group. Data are presented as median plus IQR. IQR, interquartile range; VDR, receptor of vitamin D.

Figure 2.

Expression of miRNA-346, TNF-α, and VDR in the sigmoid colon. In both patients with PSC and PSC-UC, the levels of miRNA-346 (a), TNF-α mRNA (b), VDR protein (c), and TNF-α protein (d) were comparable to the control values. In patients with UC, the miRNA-346 expression (a) was drastically suppressed, and it was accompanied by the enhanced expression of mRNA TNF-α (b), VDR protein (c), and TNF-α protein (d). MicroRNA-191 served as reference microRNA. Results are representative of n = 10 independent experiments per group. Data are presented as median plus IQR. IQR, interquartile range; PSC, primary sclerosing cholangitis; TNF-α, tumor necrosis factor-α; VDR, receptor of vitamin D.

Figure 3.

Immunohistochemical localization of VDR and TNF-α proteins in human intestinal tissue. Representative immunostaining of colonic biopsies from controls, patients with PSC, patients with PSC-UC, and patients with UC with anti-TNF-α antibodies (a–d and I–l) and anti-VDR antibodies (e–h and m–p). Brown staining indicates either VDR protein (red arrows), which is located typically in the epithelial cells, or TNF-α protein (black arrows), which is depicted in expanded apical portions of goblet cells. Nuclei were visualized by hematoxylin. Original magnification was 200×. PSC, primary sclerosing cholangitis; TNF-α, tumor necrosis factor-α; VDR, receptor of vitamin D.

Figure 4.

Effect of miR-346 inhibition or activation on TNF-α and VDR expression in cell lines. Caco-2 cells were untransfected (control) or transfected with miR-346 inhibitor (a, b) or miRNA-346 mimic (c). Twenty-four hours after transfection, levels of miR-346, TNF-α, and VDR transcript were quantified by qRT-PCR. Suppression of miR-346 by miR-346 antisense molecules was confirmed in both cell lines. Inhibition of miR-346 led to the strong upregulation of TNFα and VDR in both HepG2 (a) and Caco-2 (b) cell lines. Induction of miR-346 led to a significant downregulation of TNFα, VDR, and p27^kip1 in Caco-2 cells (c). Each experiment was repeated 4 times with similar results. Data are presented as mean ± SE. TNF-α, tumor necrosis factor-α; VDR, receptor of vitamin D.