miR-200b inhibits TGF-β1-induced epithelial-mesenchymal transition and promotes growth of intestinal epithelial cells

Abstract

Inflammatory bowel disease (IBD), which consists of Crohn's disease (CD) and ulcerative colitis (UC), is a chronic, inflammatory disorder of the gastro-intestinal tract with unknown etiology. Current evidence suggests that intestinal epithelial cells (IECs) is prominently linked to the pathogenesis of IBD. Therefore, maintaining the intact of epithelium has potential roles in improving pathophysiology and clinical outcomes of IBD. MicroRNAs (miRNAs) act as post-transcriptional gene regulators and regulate many biological processes, including embryonal development, cell differentiation, apoptosis and proliferation. In this study, we found that miR-200b decreased significantly in inflamed mucosa of IBD, especially for UC, when compared with their adjacent normal tissue. Simultaneously, we also found that the genes of E-cadherin and cyclin D1 were reduced significantly and correlated positively to the miR-200b. In addition, the upregulation of transforming growth factor-beta 1 (TGF-β1) was inversely correlated to the miR-200b in IBD. To investigate the possible roles of miR-200b in IECs maintaining, we used TGF-β1 to induce epithelial-mesenchymal transition (EMT) in IEC-6 initially. After sustained over-expressing miR-200b in IEC-6, the EMT was inhibited significantly that was characterized by downregulation of vimentin and upregulation of E-cadherin. Furthermore, we found that miR-200b enhanced E-cadherin expression through targeting of ZEB1, which encode transcriptional repressors of E-cadherin. SMAD2 was found to act as a target of miR-200b with direct evidence that miR-200b binding to the 3' UTR of SAMD2 and the ability of miR-200b to repress SMAD2 protein expression. With SMAD2 depletion, the expression of vimentin decreased correspondingly, which suggested miR-200b might reduce vimentin through regulating the SMAD2. With endogenous over-expression of miR-200b, the proliferation of IEC-6 cells increased significantly by increasing S-phase entry and promoting expression of the protein cyclin D1. Summarily, our study suggested a potential role for mir-200b in maintaining intact of intestinal epithelium through inhibiting EMT and promoting proliferation of IECs.

Figure 1

miR-200b was down-regulated in IBD. The expression of mir-200b decreased by about twofold in 22 IBD tissues when compared with the matched controls. In Ulcerative Colitis (UC, N=11) miR-200b was reduced significantly in inflamed mucosa. However, there was no significant changes in Crohn's Disease (CD, N=11). In patients with colonic adenoma, acting as the non-IBD controls, it neither found any significant changes of miR-200b in lesion of colon when compared with adjacent normal controls. *P<0.05; **P<0.01

Figure 2

The miR-200b was significantly correlated with E-cadherin in IBD. The expression of CDH1 (gene of E-cadherin) was reduced significantly in inflamed mucosa of IBD, especially in UC. Pearson correlation and linear regression analysis indicated that miR-200b was correlated significantly to CDH1 (R=0.47, i=0.02). Conversely, the gene of Vimentin increased in lesion tissues of IBD, but had no significant relationship with miR-200b (R=−0.15, P=0.36). *P<0.05, **P<0.01

Figure 3

miR-200b was correlated positively with CCND1 and negatively with TGF-β1 in IBD. As markers of cell proliferation, the genes of CCND1 and PCNA were detected and quantified. It found that CCND1 was downregulated significantly in IBD. Pearson correlation and linear regression analysis showed that CCND1 was correlated significantly to miR-200b. TGF-β1 was upregulated significantly and correlated negatively with miR-200b. *P<0.05, **P<0.01

Figure 4

The expression of miR-200b was inhibited by TGF-β1. In respond to stress of TGF-β1, the expression of miR-200b was reduced significantly in both lentiviral-vector and lentiviral-miR-200b infected cells. (a) The cell-cell junction disappeared and was became scatter in respond to the TGF-β1 including, while, miR-200b protected these cells from losing polarity. (b) *P<0.05, **P<0.01. Magnified × 100

Figure 5

miR-200b inhibited TGF-β1-induced EMT. Western-blot and immunochemistry analysis showed that TGF-β1 could induce EMT in IEC-6 cells by repression of E-cadherin expression, and increased expression of vimentin at the indicated time. With miR-200b over-expressing in IEC-6 cells, this process of EMT was inhibited significantly (a). Immunochemistry analysis indicated that showed endogenous over-expression of miR-200b decreased the vimentin and increased the E-cadherin at the seventh day of TGF-β1 inducing. (b) Arrows indicated the staining of E-cadherin or vimentin, Magnified × 200

Figure 6

MiRNA-200b targeted SMAD2 and modulated response to TGF-β1.The putative binding site of miR-200b at position of 334-340 in SMAD2 3′-UTR region was predicted by TargetScan 6.2 (http://www.targetscan.org). Expression of the firefly luciferase reporter activity was significantly reduced by sustained expression of miR-200b. Western-blot analysis also showed that the expression of SMAD2 protein decreased significantly by endogenous over-expression of miR-200b. (a) After SMAD2 knocked down, the expression of vimentin was decreased evidently. The over-expression of miR-200b inhibited expression of ZEB1 significantly. (b)

Figure 7

miR-200b promoted the proliferation of IECs. The growth of IEC-6 was suppressed by TGF-β1. While, with miR-200b over-expressing in IEC-6 cells, the proliferation was promoted significantly. (a) Cell-cycle analysis showed that cells infected with lentiviral-miR-200b had increased numbers of cells in S phase and corresponding decreased numbers of cells in G1 phase. (b) miR-200b upregulated the expression of Cyclin D1. (c) *P<0.05; **P<0.01