ABSTRACT
BACKGROUND: Trefoil factor family 1 (TFF1) is a member of the TFF-domain peptide family involved in epithelial restitution and cell motility. Recently, we screened Piezo1 as a candidate TFF1-binding protein. AIM: We aimed to confirm Piezo1 as a novel TFF1 binding protein and to assess the role of this interaction in mediating gastric cancer cell mobility. METHODS: This interaction was confirmed by co-immunoprecipitation and co-localisation of TFF1 and Piezo1 in GES-1 cells. We used stable RNA interference to knockdown Piezo1 protein expression and restored the expression of TFF1 in the gastric cancer cell lines SGC-7901 and BGC-823. Cell motility was evaluated using invasion assay and migration assay in vitro. The expression levels of the integrin subunits ß1, ß5, α1 as well as the expression of ß-catenin and E-cadherin were detected by Western blot. RESULTS: We demonstrate that TFF1, but not TFF2 or TFF3, bind to and co-localize with Piezo1 in the cytoplasm in vitro. TFF1 interacts with the C-terminal portion of the Piezo1 protein. Wound healing and trans-well assays demonstrated that the restored expression of TFF1 promoted cell mobility in gastric cancer cells, and this effect was attenuated by the knockdown of Piezo1. Western blots demonstrated the decreased expression of integrin ß1 in Piezo1-knockdown cells. CONCLUSIONS: Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.
Subject(s)
Biomarkers, Tumor/metabolism , Carrier Proteins/metabolism , Cell Movement/physiology , Ion Channels/metabolism , Stomach Neoplasms/physiopathology , Tumor Suppressor Proteins/metabolism , Blotting, Western , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Immunoprecipitation , Stomach Neoplasms/metabolism , Trefoil Factor-1 , Trefoil Factor-2ABSTRACT
Increasing evidence indicates that in gastric epithelial cells, induction of TFF3 by hypoxia is mediated by HIF-1. Since VEGF is one of the most important angiogenic factors on cancer progression, we have started to investigate the possible link among HIF-1α, VEGF, and TFF3 in gastric cancer cells. We induced the hypoxic condition in SGC-7901cells using hypoxia-mimetic agent of CoCI2. SGC7901 cells were transfected with pcPUR + U6 plasmid carrying RNAi targeted to human TFF3 and selected puromycin-resistant pools to establish the stable knockdown of TFF3 cells. Our results showed the induction of HIF-1a via hypoxia and consequences of increased expressions of the TFF3 and VEGF in gastric cancer SGC-7901 cells. Overexpression of TFF3 upregulated the mRNA expressions of VEGF and HIF-1a induced by hypoxia, and stable knockdown of TFF3 impaired the mRNA upregulations of VEGF and HIF-1a induced by hypoxia. Furthermore, knockdown of TFF3 reduced the VEGF protein secretion: as VEGF secretion was increased time dependent manner in response to the hypoxia induction in TFF3-WT cells; however, VEGF production was significantly decreased in TFF3-KD cells (621 ± 89 vs. 264 ± 73 at 6 h and 969 ± 97 vs. 508 ± 69 at 12 h, P < 0.05). Our data demonstrated the TFF3 mediated regulation of VEGF expression induced by hypoxia, and implicated that TFF3 might be applied as a potential anti-angiogenic target for treatment of gastric cancer.
Subject(s)
Gene Expression Regulation, Neoplastic , Peptides/metabolism , Stomach Neoplasms/genetics , Vascular Endothelial Growth Factor A/genetics , Cell Hypoxia/genetics , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Peptides/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trefoil Factor-3 , Up-Regulation/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
OBJECTIVE: To summarize the latest developments in silk protein fiber as biomaterials and their applications in tissue engineering. METHODS: Recent original literature on silk protein fiber as biomaterials were reviewed, illustrating the properties of silk protein fiber biomaterials. RESULTS: The silk protein fiber has the same functions of supporting the cell adhesion, differentiation and growth as native collagen, and is renewed as novel biomaterials with good biocompatibility, unique mechanical properties and is degradable over a longer time. CONCLUSION: Silk protein fiber can be used as a suitable matrix for three dimensional cell culture in tissue engineering. It has a great potential applications in other fields.
Subject(s)
Biocompatible Materials , Fibroins , Tissue Engineering , Animals , Bombyx , Cell Adhesion , Cell Division , Collagen/chemistry , Extracellular Matrix/chemistry , Fibroins/chemistry , Fibroins/metabolism , Insect Proteins , SilkABSTRACT
Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity. Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed. DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG. The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L. The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90%. The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD. This value and amino acid analysis were consistent with those of theoretic calculation.
