Long non-coding RNA-ATB promotes EMT during silica-induced pulmonary fibrosis by competitively binding miR-200c

Long non-coding RNAs (lncRNAs) are important signal transduction regulators that act by various patterns. However, little is known about the molecular mechanisms of lncRNA related pathways in occupational lung fibrosis. Our previous study found that epithelial-mesenchymal transition (EMT) was one of the key events in silica-induced pulmonary fibrosis. This study showed that the lncRNA-ATB promoted EMT by acting as a miR-200c sponge. miR-200c was identified by miRNA array as a potential target of lncRNA-ATB and verified by dual luciferase reporter gene together with RNA pull-down assays. Moreover, our findings demonstrated that lncRNA-ATB is abundantly expressed during EMT of lung epithelial cells, which contributes to decreased levels of miR-200c. miR-200c targeted ZEB1 to relief silicosis by blocking EMT in vivo and in vitro. The results also suggested M2 macrophages secreted transforming growth factor-β1 (TGF-β1) to induce EMT process by activating lncRNA-ATB in epithelial cells. Collectively, silica-stimulated macrophages secreted TGF-β1 to induce lncRNA-ATB in epithelia cells, promoting EMT by binding with miR-200c and releasing ZEB1. These observations provide further understanding of the regulatory network of silica-induced pulmonary fibrosis and identify new therapeutic targets hopefully.     

Coupled Reversible and Irreversible Bistable Switches Underlying TGFβ-induced Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) plays an important role in embryonic development, tissue regeneration, and cancer metastasis. Whereas several feedback loops have been shown to regulate EMT, it remains elusive how they coordinately modulate EMT response to TGF-β treatment. We construct a mathematical model for the core regulatory network controlling TGF-β-induced EMT. Through deterministic analyses and stochastic simulations, we show that EMT is a sequential two-step program in which an epithelial cell first is converted to partial EMT then to the mesenchymal state, depending on the strength and duration of TGF-β stimulation. Mechanistically the system is governed by coupled reversible and irreversible bistable switches. The SNAIL1/miR-34 double-negative feedback loop is responsible for the reversible switch and regulates the initiation of EMT, whereas the ZEB/miR-200 feedback loop is accountable for the irreversible switch and controls the establishment of the mesenchymal state. Furthermore, an autocrine TGF-β/miR-200 feedback loop makes the second switch irreversible, modulating the maintenance of EMT. Such coupled bistable switches are robust to parameter variation and molecular noise. We provide a mechanistic explanation on multiple experimental observations. The model makes several explicit predictions on hysteretic dynamic behaviors, system response to pulsed stimulation, and various perturbations, which can be straightforwardly tested.     

Growth factor TGF-β induces intestinal epithelial cell (IEC-6) differentiation: miR-146b as a regulatory component in the negative feedback loop

TGF-β is a potent pleiotropic factor that promotes small intestinal cell differentiation. The role of microRNAs in the TGF-β induction of intestinal epithelial phenotype is largely unknown. We hypothesized that microRNAs are functionally involved in TGF-β-induced intestinal cell growth. In this study, TGF-β caused a morphological change of IEC-6 cells and stimulated expression of the epithelial cell markers alkaline phosphatase, villin, and aminopeptidase N. By global microRNA profiling during TGF-β-induced intestinal crypt cell (IEC-6) differentiation, we identified 19 differentially expressed microRNAs. We showed by real-time Q-PCR that miR-146b expression increased rapidly after TGF-β treatment; sequence analysis and in vitro assays revealed that miR-146b targets SIAH2, an E3 ubiquitin ligase, with decreased protein expression upon IEC-6 cell differentiation. Transfection of miR-146b inhibitor before TGF-β treatment blocked the down-regulation of SIAH2 in response to TGF-β. Moreover, SIAH2 over-expression during TGF-β treatment caused a significant decrease in Smad7 protein expression in IEC-6 cells. Furthermore, activation of the ERK1/2 pathway is active in the up-regulation of miR-146b by TGF-β. These findings suggest a novel mechanism whereby TGF-β signaling during IEC-6 cell differentiation may be modulated in part by microRNAs, and we propose a key role for miR-146b in the homeostasis of growth factor TGF-β signaling through a negative feedback regulation involving down-regulation of SIAH2 repressed Smad7 activities.     

MIR663AHG as a competitive endogenous RNA regulating TGF-β-induced epithelial proliferation and epithelial–mesenchymal transition in benign prostate hyperplasia

Benign prostate hyperplasia (BPH) is the most commonly seen disease among aging males. Transforming growth factor(TGF)-β-mediated epithelial–mesenchymal transition (EMT) and epithelial overproliferation might be central events in BPH etiology and pathophysiology. In the present study, long noncoding RNA MIR663AHG, miR-765, and FOXK1 formed a competing endogenous RNAs network, modulating TGF-β-mediated EMT and epithelial overproliferation in BPH-1 cells. miR-765 expression was downregulated in TGF-β-stimulated BPH-1 cells; miR-765 overexpression ameliorated TGF-β-mediated EMT and epithelial overproliferation in BPH-1 cells. MIR663AHG directly targeted miR-765 and negatively regulated miR-765; MIR663AHG knockdown also attenuated TGF-β-induced EMT and epithelial overproliferation in BPH-1 cells, whereas miR-765 inhibition attenuated MIR663AHG knockdown effects on TGF-β-stimulated BPH-1 cells. miR-765 directly targeted FOXK1 and negatively regulated FOXK1. FOXK1 knockdown attenuated TGF-β-induced EMT and epithelial overproliferation and promoted autophagy in BPH-1 cells, and partially attenuated miR-765 inhibition effects on TGF-β-stimulated BPH-1 cells. In conclusion, this study provides a MIR663AHG/miR-765/FOXK1 axis modulating TGF-β-induced epithelial proliferation and EMT, which might exert an underlying effect on BPH development and act as therapeutic targets for BPH treatment regimens.     

Attenuation of TGF-β signaling suppresses premature senescence in a p21-dependent manner and promotes oncogenic Ras-mediated metastatic transformation in human mammary epithelial cells

The molecular mechanisms that drive triple-negative, basal-like breast cancer progression are elusive. Few molecular targets have been identified for the prevention or treatment of this disease. Here we developed a series of isogenic basal-like human mammary epithelial cells (HMECs) with altered transforming growth factor-β (TGF-β) sensitivity and different malignancy, resembling a full spectrum of basal-like breast carcinogenesis, and determined the molecular mechanisms that contribute to oncogene-induced transformation of basal-like HMECs when TGF-β signaling is attenuated. We found that expression of a dominant-negative type II receptor (DNRII) of TGF-β abrogated autocrine TGF-β signaling in telomerase-immortalized HMECs and suppressed H-Ras-V12-induced senescence-like growth arrest (SLGA). Furthermore, coexpression of DNRII and H-Ras-V12 rendered HMECs highly tumorigenic and metastatic in vivo in comparison with H-Ras-V12-transformed HMECs that spontaneously escaped H-Ras-V12-induced SLGA. Microarray analysis revealed that p21 was the major player mediating Ras-induced SLGA, and attenuated or loss of p21 expression contributed to the escape from SLGA when autocrine TGF-β signaling was blocked in HMECs. Furthermore, knockdown of p21 also suppressed H-Ras-V12-induced SLGA. Our results identify that autocrine TGF-β signaling is an integral part of the cellular anti-transformation network by suppressing the expression of a host of genes, including p21-regulated genes, that mediate oncogene-induced transformation in basal-like breast cancer.     

Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl₄-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.     

Activation of Wnt11 by transforming growth factor-β drives mesenchymal gene expression through non-canonical Wnt protein signaling in renal epithelial cells

Transforming growth factor β1 (TGF-β) promotes renal interstitial fibrosis in vivo and the expression of mesenchymal genes in vitro; however, most of its direct targets in epithelial cells are still elusive. In a screen for genes directly activated by TGF-β, we found that components of the Wnt signaling pathway, especially Wnt11, were targets of activation by TGF-β and Smad3 in primary renal epithelial cells. In gain and loss of function experiments, Wnt11 mediated the actions of TGF-β through enhanced activation of mesenchymal marker genes, such as Zeb1, Snail1, Pai1, and αSMA, without affecting Smad3 phosphorylation. Inhibition of Wnt11 by receptor knockdown or treatment with Wnt inhibitors limited the effects of TGF-β on gene expression. We found no evidence that Wnt11 activated the canonical Wnt signaling pathway in renal epithelial cells; rather, the function of Wnt11 was mediated by the c-Jun N-terminal kinase (JNK) pathway. Consistent with the in vitro results, all the TGF-β, Wnt11, and JNK targets were activated in a unilateral ureteral obstruction (UUO) model of renal fibrosis in vivo. Our findings demonstrated cooperativity among the TGF-β, Wnt11, and JNK signaling pathways and suggest new targets for anti-fibrotic therapy in renal tissue.