uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.
␣-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of ␣-globin protein. Couples who both carry the ␣-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the ␣-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study , a new and accurate method for ␣-thalassemia diagnosis was developed by genotyping ␣-thalassemia-1, the Southeast Asian type (؊؊ SEA ) and Thai type (؊؊ THAI ) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments , the ؊؊ SEA and ؊؊ THAI deletions and two normal fragments , -and ␣2-globin gene. The primers were capable of distinguishing ␣-thalassemia 1 heterozygotes from ␣-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the ؊؊ THAI , ؊؊ SEA , -globin, and ␣2-globin gene fragments were 79.9 ؎ 0.2 , 89.4 ؎ 0.5, 92.8 ؎ 0.2 , and 85.0 ؎ 0.2°C , respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis , and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process , which is laborious , and allows high throughput screening suitable for large population screening for prevention and control
BackgroundTransforming growth factor-β (TGF-β) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-β tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells.MethodsTGF-β1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-β1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells.Resultsh-TGF-β1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine’s ability to attenuate cell proliferation. h-TGF-β1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-β1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-β-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-β1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knock-down suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-β1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-β1 tumor promoting but repressed its tumor suppressing functions.ConclusionsInhibiting ERK1/2 activation attenuates TGF-β1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-β paradox switch.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-017-0454-2) contains supplementary material, which is available to authorized users.
Extensive desmoplasia in cholangiocarcinoma (CCA) is associated with tumor aggressiveness, indicating a need for further understanding of CCA cell-matrix interaction. This study demonstrated laminin as the most potent attractant for CCA cell migration and the vast elevation of its receptor integrin β4 (ITGB4) in CCA cell lines. Besides, their high expressions in CCA tissues were correlated with lymphatic invasion and the presence of ITGB4 was also associated with short survival time. ITGB4 silencing revealed it as the receptor for laminin-induced HuCCA-1 migration, but KKU-213 utilized 37/67-kDa laminin receptor (LAMR) instead. These findings highlight the role of ITGB4 and LAMR in transducing laminin induction of CCA cell migration and the potential of ITGB4 as diagnostic and prognostic biomarkers for CCA.
Cell sheet engineering, a scaffold-free tissue fabrication technique, has proven to be an important breakthrough technology in regenerative medicine. Over the past two decades, the field has developed rapidly in terms of investigating fabrication techniques and multipurpose applications in regenerative medicine and biological research. This review highlights the most important achievements in cell sheet engineering to date. We first discuss cell sheet harvesting systems, which have been introduced in temperature-responsive surfaces and other systems to overcome the limitations of conventional cell harvesting methods. In addition, we describe several techniques of cell sheet transfer for preclinical (in vitro and in vivo) and clinical trials. This review also covers cell sheet cryopreservation, which allows short- and long-term storage of cells. Subsequently, we discuss the cell sheet properties of angiogenic cytokines and vasculogenesis. Finally, we discuss updates to various applications, from biological research to clinical translation. We believe that the present review, which shows and compares fundamental technologies and recent advances in cell engineering, can potentially be helpful for new and experienced researchers to promote the further development of tissue engineering in different applications.
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