miR-448通过抑制上皮间质转化抑制肺癌细胞增殖和运动能力

探讨mi R-448对肺癌细胞增殖和运动的影响及其分子机制。采用实时荧光定量PCR(polymerase chain reaction)检测原发肺癌组织和癌旁正常组织mi R-448表达水平。转染mi R-448 mimic和inhibitor至肺癌A549细胞系,通过MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide)、平板克隆形成和Transwell实验观察mi R-448表达对A549增殖和运动能力的影响;利用Western blot检测EMT(epithelial-mesenchymal transition)标志物蛋白表达水平,通过实时荧光定量PCR检测EMT相关转录因子m RNA表达水平。实时荧光定量PCR显示较癌旁正常组织相比,mi R-448在原发肺癌组织中表达降低。MTT和平板克隆形成实验显示,过表达mi R-448抑制A549细胞增殖和运动能力;降表达mi R-448增强A549细胞增殖和运动能力。Western blot显示降表达mi R-448能下调上皮标志物E-cadherin,上调间质标志物Vimentin表达水平。实时荧光定量PCR显示降表达mi R-448能上调EMT相关转录因子Twist1和ZEB1 m RNA表达水平。mi R-448可通过抑制EMT抑制肺癌进展。     

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.     

Inhibition of Spiralin Processing by the Lipopeptide Antibiotic Globomycin

The cyclic lipopeptide globomycin, a specific inhibitor of signal-peptidase II (Lsp A), proved toxic for the mollicute Spiroplasma melliferum with a minimal inhibitory concentration (MIC) in the range 6.25–12.5 μM, about one order of magnitude higher (that is, less efficient) than bee-venom mellitin. SDS-PAGE analysis of cell proteins followed by immunolabeling (“Western blotting”) and by crossed immunoelectrophoresis demonstrated that the cleavage of the prespiralin leader peptide was prevented by globomycin. Cell fractionation experiments showed that prespiralin was membrane bound and did not accumulate in the cytoplasm or in the culture medium. Furthermore, the use of the potential-sensitive fluorescent dye 3,3′-dipropyl-2,2′-thiadicarbocyanine iodide (diS-C₃-[5]) revealed that, in contrast to valinomycin and mellitin, globomycin up to 30 μM has no effect on the electrical transmembrane potential of S. melliferum. This indicates that the toxicity of globomycin for spiroplasma cells is mainly if not exclusively owing to the inhibition of spiralin processing. Added to previously published data, these results suggest that spiralin and probably other lipoproteins of mollicutes are acylated and membrane targeted by a mechanism involving notably the processing of the prelipoprotein precursor by a type II, globomycin-sensitive signal peptidase. Received: 19 April 1996 / Accepted: 28 May 1996     

Goblet Cell Increase in Rat Bronchial Epithelium after Exposure to Cigarette and Cigar Tobacco Smoke

Both cigar and cigarette tobacco produce an increase in the number of goblet cells in the rat trachea and intrapulmonary airways over a six-week period. The increase in goblet cells is similar for the two types of tobacco; in both it is proportional to increase in dose and greatest in the proximal intrapulmonary airways.     

Tamoxifen Ameliorates Peritoneal Membrane Damage by Blocking Mesothelial to Mesenchymal Transition in Peritoneal Dialysis

Mesothelial-to-mesenchymal transition (MMT) is an auto-regulated physiological process of tissue repair that in uncontrolled conditions such as peritoneal dialysis (PD) can lead to peritoneal fibrosis. The maximum expression of peritoneal fibrosis induced by PD fluids and other peritoneal processes is the encapsulating peritoneal sclerosis (EPS) for which no specific treatment exists. Tamoxifen, a synthetic estrogen, has successfully been used to treat retroperitoneal fibrosis and EPS associated with PD. Hence, we used in vitro and animal model approaches to evaluate the efficacy of Tamoxifen to inhibit the MMT as a trigger of peritoneal fibrosis. In vitro studies were carried out using omentum-derived mesothelial cells (MCs) and effluent-derived MCs. Tamoxifen blocked the MMT induced by transforming growth factor (TGF)-β1, as it preserved the expression of E-cadherin and reduced the expression of mesenchymal-associated molecules such as snail, fibronectin, collagen-I, α-smooth muscle actin, and matrix metalloproteinse-2. Tamoxifen-treatment preserved the fibrinolytic capacity of MCs treated with TGF-β1 and decreased their migration capacity. Tamoxifen did not reverse the MMT of non-epitheliod MCs from effluents, but it reduced the expression of some mesenchymal molecules. In mice PD model, we demonstrated that MMT progressed in parallel with peritoneal membrane thickness. In addition, we observed that Tamoxifen significantly reduced peritoneal thickness, angiogenesis, invasion of the compact zone by mesenchymal MCs and improved peritoneal function. Tamoxifen also reduced the effluent levels of vascular endothelial growth factor and leptin. These results demonstrate that Tamoxifen is a therapeutic option to treat peritoneal fibrosis, and that its protective effect is mediated via modulation of the MMT process.     

Collagen XV Inhibits Epithelial to Mesenchymal Transition in Pancreatic Adenocarcinoma Cells

Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.     

上皮间质转化标记物及Snail在子宫内膜异位症中的表达

目的:研究上皮间质转化标志物(E-cadherin、β-catenin、vimentin)和Snail在子宫内膜异位症(endometriosis,EMs)中的表达。方法:选取40例EMs患者(实验组)异位内膜及在位内膜,同时获取20例非EMs患者(对照组)的正常子宫内膜,采用免疫组化法研究Snail、EMT上皮标志物(E-cadherin、β-catenin)、间质标志物(vimentin)在各内膜组织中的表达,并比较其表达水平。结果:EMs患者异位内膜和在位内膜的EMT上皮标志物E-cadherin、β-catenin表达均显著低于正常内膜的表达(P0.05);EMs患者异位内膜和在位内膜的EMT间质标志物vimentin表达均显著高于正常内膜的表达(P0.05);EMs患者异位内膜和在位内膜中Snail表达显著高于正常内膜的表达(P0.05)。结论:在子宫内膜异位症(EMs)中,Snail、vimentin表达上调,E-cadherin、β-catenin表达下调可能与子宫内膜异位症(EMs)的发生、发展及浸润转移有关。     

Selective Inhibition of Type II Fatty Acid Synthetase by the Antibiotic Thiolactomycin

The antibiotic thiolactomycin inhibits the fatty acid synthesisfrom both [1-¹⁴C]- acetate and [2-¹⁴C]malonyl-CoA of spinachleaves, developing castor bean endosperms and avocado mesocarp.On the other hand, fatty acid synthetases of Brevibacteriumammoniagenes and Corynebacterium glutamicum are much less sensitiveto this antibiotic. As has been indicated that thiolactomycininhibits fatty acid synthetase of Escherichia coli but has littleeffect on the synthetases of yeast and rat liver [Hayashi etal. (1983) Biochem. Biophys. Res. Commun.. 115: 1108], thiolactomycinis suggested to be a selective inhibitor of type II fatty acidsynthetases. (Received November 10, 1983; Accepted December 17, 1983)     

Transdifferentiation of Pigmented Epithelium Induced by the Influence of Lens Epithelium in Frogs

Abstract. The influence of lens epithelium (LE) of adult frogs on the character of transdifferentiation of retinal pigmented epithelium (RPE) of adult frogs and tadpoles of Rana temporaria has been studied. After a period of intense proliferation RPE cultured in vivo in contact with LE in the tadpole orbit almost exclusively transforms into retina. RPE precultivated in vitro in contact with LE for three days in protein-free medium does not manifest cell divisions and mostly transdifferentiates into lentoids. The problem of the relative significance of inducing determinants and the role of activation or inhibition of proliferation in transdifferentiation is discussed.     

A Biophysical Model for Integration of Electrical, Osmotic, and pH Regulation in the Human Bronchial Epithelium

A dynamical biophysical model for the functioning of an epithelium is presented. This model integrates the electrical and osmotic behaviors of the epithelium, taking into account intracellular conditions. The specific tissue modeled is the human bronchial epithelium, which is of particular interest, as it is the location of the most common lethal symptoms of cystic fibrosis. The model is implemented in a modular form to facilitate future application of the code to other epithelial tissue by inputting different transporters, channels, and geometric parameters. The model includes pH regulation as an integral component of overall regulation of epithelial function, through the interdependence of pH, bicarbonate concentration, and current. The procedures for specification, the validation of the model, and parametric studies are presented using available experimental data of cultured human bronchial epithelium. Parametric studies are performed to elucidate a), the contribution of basolateral chloride channels to the short-circuit current functional form, and b), the role that regulation of basolateral potassium conductance plays in epithelial function.