Dickkopf-1 silencing inhibits invasion and epithelial-mesenchymal transition in gastric carcinoma cells by down-regulating β-catenin

AIM: To explore the expression of Dickkopf-1 (DKK1) in human gastric carcinoma cells, and the influences of DKK1 gene silencing on cell invasion. METHODS: The levels of DKK1 in the human gastric mucosa cell line GES-1 and gastric carcinoma cell lines MKN-45 and SGC-7901 were detected by real-time PCR and Western blot. DKK1 gene was silenced by RNA interference, which was verified by real-time PCR, Western blot and ELISA. The cell invasion ability was determined by Transwell assay, and the cell proliferation was inhibited by mitomycin C. The levels of E-cadherin, N-cadherin, vimentin and β-catenin were determined by real-time PCR and Western blot. RESULTS: The expression of DKK1 was significantly higher in MKN-45 cells and SGC-7901 cells than that in GES-1 cells, indicating that DKK1 expression was obviously increased in gastric carcinoma cells. After successful silencing of DKK1 gene in the MKN-45 cells and SGC-7901 cells, the cell invasion ability was markedly decreased in a time-dependent pattern with increased expression of E-cadherin and decreased expression of N-cadherin and vimentin, indicating that DKK1 silencing dramatically inhibited gastric carcinoma cell invasion and epithelial-mesenchymal transition (EMT). The introduction of exogenous recombinant DKK1 (rDKK1) demonstrated the promoting effect of DKK1 on gastric carcinoma cell invasion and EMT. In addition, the inhibitory effects of DKK1 silencing on gastric carcinoma cell invasion and EMT were fulfilled by down-regulating β-catenin. CONCLUSION: The expression of DKK1 is significantly increased in human gastric carcinoma cells. Silencing of DKK1 markedly inhibits gastric carcinoma cell invasion and EMT by down-regulating β-catenin.     

Effect of hirsutine on hypoxia-induced migration and invasion abilities in human breast cancer MCF-7 cells

AIM: To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS: CCK-8 assay was employed to detect the cytotoxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay, and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS: Hirsutine remarkably reduced the cell viability from 32 μmol/L (P<0.05), and the IC₅₀ value was 62.82 μmol/L. In hypoxia state, MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α, Snail and MMP-9 (P<0.05), lower protein level of E-cadherin (P<0.05), and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05), apart from the mRNA level of HIF-1α. CONCLUSION: Hirsutine inhibits hypoxia-induced migration and invasion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α, Snail and MMP-9, and up-regulation of the protein level of E-cadherin.     

Reverse effect of FOXC2 silencing on epithelial-mesenchymal transition induced by TGF-β1 in MCF-7 cells

AIM: To study the reverse effect of FOXC2 silencing on epithelial-mesenchymal transition (EMT) induced by transforming growth factor β1(TGF-β1) in MCF-7 cells. METHODS: Cultured MCF-7 cells were treated with TGF-β1 at concentration of 5 μg/L for 6 d. The cell morphological changes were observed under phase-contrast microscope. The changes of EMT-related marker proteins were assessed by immunofluorescence staining assay. TGF-β1-induced MCF-7 cells were transfected with FOXC2-siRNA mediated by recombinant lentivirus. In addition, the expression levels of FOXC2 and EMT-related marker proteins E-cadherin, claudin-1 and fibronectin-1 were also measured by RT-PCR and Western blotting. The invasion of MCF-7 cells was detected by Transwell assay. RESULTS: TGF-β1 induced the morphological alteration in MCF-7 cells from epithelial phenotype to mesenchymal phenotype,up-regulated the expression of mesenchymal marker fibronectin-1, and down-regulated the expression of epithelial markers E-cadherin and claudin-1. FOXC2 silencing reversed and restored the mesenchymal MCF-7 cells to epithelial phenotype and reduced the tumor invasion. CONCLUSION: EMT model induced by TGF-β1 in breast cancer MCF-7 cells is successfully established, which increases the invasion of MCF-7 cells. The effect of TGF-β1 is reversed by FOXC2-siRNA and the invasion of the cells is reduced.     

Effect of luteolin on TGF-β1-induced epithelial-mesenchymal transition in lung cancer A549 cells

AIM:To investigate the effects of luteolin on the invasion and epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1) in lung cancer A549 cells. METHODS:The effect of luteolin at 5, 10, 20, 40, 80 and 160 μmol/L on the viability of A549 cells was measured by MTT assay. The invasion ability was analyzed by Transwell method. The morphological changes of the A549 cells were observed under microscope.The protein expression of E-cadherin and vimentin in the A549 cells were determined by Western blot. RESULTS:The viability of the A549 cells was significantly inhibited by luteolin in a dose-time dependent manner (P<0.05). The IC₅₀ of luteolin for the A549 cells (24 h) was 68.79 μmol/L, while that (48 h) was 47.86 μmol/L. TGF-β1 induced morphological alteration of the A549 cells from epithelial to mesenchymal forms. Luteolin significantly inhibited TGF-β1-induced invasion of the A549 cells (P<0.01). The protein expression of E-cadherin was significantly down-regulated and the protein expression of vimentin was significantly up-regulated in the presence of TGF-β1 at 5 μg/L (P<0.01). However, luteolin reversed TGF-β1-induced EMT, up-regulation of E-cadherin and down-regulation of vimentin (P<0.01). CONCLUSION:Lu-teolin reverses TGF-β1-induced EMT in the lung cancer A549 cells.     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Effect of toosendanin on invasion and migration of human ovarian cancer cells

AIM: To investigate the effect of toosendanin (TSN) on invasion and migration abilities of human ovarian cancer cells and the related mechanism. METHODS: The human ovarian cancer cell lines CAVO-3 and SKVO-3 were treated with TSN at different concentrations. The cell viabilty at 12, 24, 48, 72 and 96 h after TSN treatment was measured by CCK-8 assay. Scratch wound healing assay and Transwell assay were employed to measure the invasion and migration abilities of CAVO-3 cells. The protein expression of nuclear factor-κB (NF-κB) p65, E-cadherin, N-cadherin, vimentin and Snail was determined by Western blot. RESULTS: TSN significantly inhibited the viability of CAVO-3 and SKVO-3 cells (P<0.05). Compared with control group, the migration and invasion abilities of CAVO-3 cells in TSN group decreased significantly (P<0.05). In addition, the expression of NF-κB p65 and E-cadherin protein increased notably, followed with N-cadherin, vimentin and Snail protein decreased significantly (P<0.05). However, the inhibitor of NF-κB BAY11-7082 reversed the impact above. Compared with TSN group, the migration and invasion abilities in TSN+BAY11-7082 group increased significantly (P<0.05). The protein expression of E-cadherin also decreased notably, followed with the protein expression of N-cadherin, vimentin and Snail increased significantly (P<0.05). CONCLUSION: TSN inhibits the invasion and migration abilities of human ovarian cancer cells, which is related to the inhibition of epithelial-mesenchymal transition process mediated by NF-κB/Snail signaling pathway.     

Emodin inhibits proliferation and blocks cell cycle of human breast cancer MCF-7 cells

AIM：To investigate the effects of emodin on the proliferation of human breast cancer MCF-7 cells and its mechanisms. METHODS：MTT assay was used to observe the viability of MCF-7 cells. The cell cycle distribution and apoptosis of MCF-7 cells was analyzed by flow cytometry. The membrane surface morphology and three-dimensional ultrastructure of MCF-7 cells were observed under atomic force microscope (AFM). RESULTS：MTT assay showed that emodin could inhibit MCF-7 cell proliferation in a dose-dependent manner. Flow cytometric analysis demonstrated that emodin induced cell cycle arrest at G0/G1 phase. Annexin V/PI double staining confirmed that emodin had no effect on cell apoptosis. AFM images revealed that the cell nuclear area was full and the surface of cell membrane was flat and smooth in control group. Compared with control group, the cell nuclear area collapsed and shrank in emodin group at 48 h. The cell membrane ultrastructure showed that the particles in emo-din group had an intensive distribution. The height of cell nuclear area was decreased, and the surface average roughness (Ra) and root mean square roughness (Rq) were elevated in emo-din group compared with control group. CONCLUSION: Emodin has cytotoxicity on MCF-7 cells via cell cycle arrest at G0/G1 phase and ultrastructural changes.     

Effect of IGFBP7 on proliferation of human breast cancer cell line MCF-7

AIM: To investigate the mechanism that insulin-like growth factor binding protein 7 (IGFBP7) inhibits proliferation of human breast cancer cell line MCF-7. METHODS: Plasmid pCMV6-IGFBP7 or empty plasmid was transfected into MCF-7 cells. The expression of IGFBP7 in MCF-7 cells after transfection was detected by Western blotting. The effects of IGFBP7 on the colony-forming efficiency and the cell cycle were studied by soft agar colony formation assay and flow cytometry,respectively. The effects of IGFBP7 on the expression of ERK1/2, p-ERK1/2, cyclin D1, CDK4, cyclin E, CDK2, p21^CIP1/WAF1, p27^KIP1, p53, Rb and p-Rb in MCF-7 cells were detected by Western blotting. RESULTS: Only the transfectant of pCMV6-IGFBP7 expressed IGFBP7. IGFBP7 remarkably reduced colony-forming efficiency (P<0.01) and G₀/G₁ arrest (P<0.01), inhibited phosphorylation of ERK1/2 (P<0.01), down-regulated cyclin D1 and cyclin E (P<0.01), up-regulated p27^KIP1, p21^CIP1/WAF1 and p53 (P<0.01), and inhibited phosphorylation of Rb (P<0.01) in MCF-7 cells. PD98059, an inhibitor of MEK1 and MEK2, imitated part of the tumor-suppressing activity of IGFBP7. CONCLUSION: IGFBP7 inhibits the proliferation of human breast cancer cell line MCF-7 by down-regulating cyclin D1 and cyclin E, up-regulating p27^KIP1, p21^CIP1/WAF1 and p53 and inhibiting phosphorylation of Rb. ERK1/2 signaling pathway might be involved in the regulation of cyclin D1 and p27^KIP1 by IGFBP7.     

IL-6 promotes epithelial-mesenchymal transition,migration and invasion of papillary thyroid carcinoma TPC-1 cells by inducing lncTCF7 expression

AIM:To investigate the effect of interleukin-6 (IL-6) on epithelial-mesenchymal transition (EMT), migration and invasion of papillary thyroid carcinoma TPC-1 cells by inducing the expression of long noncoding RNA lncTCF7. METHODS:The effects of IL-6 on the expression of lncTCF7 in the TPC-1 cells were detected by RT-qPCR after the TPC-1 cells were treated with IL-6 at 0, 5, 10, 20 and 50 μg/L for 24 h or with IL-6 at 50 μg/L for 0, 6, 12 and 24 h. After the TPC-1 cells were treated with IL-6 at 50 μg/L for 24 h, the effect of IL-6 on the protein expression of E-cadherin and vimentin in the TPC-1 cells was detected by Western blot. The TPC-1 cell line with lncTCF7 over-expression was established, and the effects of lncTCF7 over-expression on EMT, migration and invasion of the TPC-1 cells were measured by Western blot and Transwell assay. After knockdown of lncTCF7 expression and exposure to IL-6 at 50 μg/L, the effects of lncTCF7 on EMT, migration and invasion of TPC-1 cells treated with IL-6 were observed. RESULTS:The expression of lncTCF7 in the TPC-1 cells was induced by IL-6 in a dose-and time-dependent manner. The expression of E-cadherin was down-regulated, the expression of vimentin was up-regulated, and the migration and invasion abilities of the TPC-1 cells were enhanced by lncTCF7 over-expression (P<0.05). The expression of E-cadherin was decreased, the expression of vimentin, Snail and Slug was increased, and the migration and invasion abilities of the TPC-1 cells and intercellular space were enhanced by IL-6. The above changes induced by IL-6 were significantly inhibited by knockdown of lncTCF7 expression. CONCLUSION:IL-6 promotes the EMT, migration and invasion of papillary thyroid carcinoma TPC-1 cells by inducing the expression of lncTCF7.     

MicroRNA-200a affects malignant biological behaviors of breast cancer cells by inhibiting epithelial-mesenchymal transition

AIM: To investigate the effect of microRNA-200a (miR-200a) on the malignant biological beha-viors of breast cancer cells and its regulatory mechanism. METHODS: The expression of miR-200a in human breast can-cer cell lines MDA-MB-231, MDA-MB-468 and MCF-7, and normal human mammary epithelial cell line MCF-10A was detected by RT-qPCR. CCK-8 assay was used to detect the viability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. Flow cytometry method and Transwell assay were used to detect the apoptosis and invasive ability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. The expression of SIP1, E-cadherin, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was determined by RT-qPCR and Western blot. RESULTS: Compared with MCF-10A cells, the lowest expression of miR-200a was observed in the MDA-MB-231 cells (P<0.05). Over-expression of miR-200a attenuated the viability of MDA-MB-231 cells (P<0.05), increased apoptosis (P<0.05) and decreased the invasion ability (P<0.05). The expression of SIP1, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was also significantly down-regulated, while the mRNA and protein expression of E-cadherin was significantly increased (P<0.05). Transfection with miR-200a inhibitor reversed the above results. CONCLUSION: Up-regulation of miR-200a inhibits the viability and invasion ability of MDA-MB-231 cells and promotes the apoptosis of MDA-MB-231 cells. miR-200a may regulate the biological behaviors of breast cancer by inhibiting epithelial-mesenchymal transition.     

Effect of extracellular HSP70/HSP70-PCs on epithelial-mesenchymal transition of human hepatocellular carcinoma HepG2 cells

AIM：To investigate the effect of extracellular heat-shock protein 70 (HSP70)/HSP70-peptide complexes (HSP70-PCs) on epithelial-mesenchymal transition (EMT) of human hepatocellular carcinoma HepG2 cells and its probable mechanism. METHODS：HepG2 cells were divided into 3 groups: control group, HSP70/HSP70-PCs (2 mg/L) group and LY294002+HSP70/HSP70-PCs group. The mRNA and protein expression of epithelial cell surface marker E-cadherin, mesenchymal cell surface marker α-smooth muscle actin (α-SMA), phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) was examined by real-time RT-PCR and Western blotting. RESULTS：Extracellular HSP70/HSP70-PCs promoted the initiation of EMT of HepG2 cells. The expression of HIF-1α and PI3K significantly increased in the process of EMT of HepG2 cells. After PI3K was blocked by LY294002, EMT did not occur and HIF-1α was not up-regulated in HepG2 cells. CONCLUSION：Extracellular HSP70/HSP70-PCs may promote EMT of hepatocellular carcinoma cells via PI3K/HIF-1α signaling pathway.     

Effects of luteolin on invasion,migration and adhesion of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the effects of luteolin on invasion, migration and adhesion of human hepatocelluar carcinoma HepG2 cells.METHODS: HepG2 cells were cultured and treated with luteolin at 10, 20 and 40 μmol/L respectively. The invasion capability was examined by cell invasion assay. The migration ability was examined by wound healing assay. The adhesion capability was measured by adhesion assay. The protein levels of E-cadherin, N-cadherin, vimentin and Snai1 were determined by Western blot analysis.RESULTS: Luteolin inhibited the invasion, migration and adhesion ability of HepG2 cells in vitro in a dose-dependent manner. After treatment with luteolin, the expression of E-cadherin was increased significantly and the expression of N-cadherin, vimentin and Snai1 were decreased significantly.CONCLUSION: Luteolin inhibits the invasion, migration and adhesion ability of human hepatocelluar carcinoma HepG2 cells. The mechanism may be related to the regulatory effects of luteolin on epithelial-mesenchymal transition.     

Hypoxia-induced caveolin-1 up-regulation is involved in migration and invasion of lung adenocarcinoma A549 cells

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride (CoCl2) on caveolin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcinoma A549 cells. METHODS:The concentrations of Cav-1 and hypoxia-inducible factor (HIF)-1α in pleural effusion of the patients with lung cancer (MPE) or tuberculous pleurisy (TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1α inhibitor YC-1. The concentrations of Cav-1 and HIF-1α in the cell supernatants were measured by ELISA. The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respectively. RESULTS:The levels of Cav-1 and HIF-1α in MPE were significantly higher than those in TBPE. There was a highly positive correlation between Cav-1 and HIF-1α levels in the pleural effusion. CoCl2 induced the generation of Cav-1 and HIF-1α in A549 cells in a concentration- and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration- or time-dependent inhibition was observed. HIF-1α inhibitor YC-1 concentration-dependently inhibited the generation of HIF-1α and Cav-1 induced by CoCl2 in A549 cells. CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1. CONCLUSION: The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells. A possible role for HIF-1α is indicated in Cav-1 generation.     

Over-expression of SATB1 mediates invasion and metastasis of nasopharyngeal carcinoma cells through epithelial-mesenchymal transition

AIM:To analyze the high expression of special AT-rich sequence-binding protein 1 (SATB1) in nasopharyngeal carcinoma (NPC) and its role in tumor invasion and metastasis. METHODS:The method of immunohistochemistry was used to detect the expression of SATB1 and epithelial-mesenchymal transition (EMT)-related molecules E-cadherin and vimentin in 76 cases of NPC and 61 cases of nasopharyngeal chronic inflammation (NPI), and the correlations of over-expression of SATB1 with NPC patients' clinical parameters as well as the expression of E-cadherin and vi-mentin were analyzed. Variously differentiated NPC cell lines CNE1, CNE2Z and C666-1 were cultured in vitro, and then SATB1-overexpressing cell line was screened. After interfering with SATB1 expression by siRNA, the expression of EMT-related molecules and the change of cell invasiveness were analyzed. RESULTS:The expression of SATB1 in the nasopharyngeal tissue was dominantly localized in the nuclei. The positive rate of SATB1 in NPC group was significantly higher than that in NPI group (P<0.01). E-cadherin was membrane-positive in NPI epithelial cells, while membrane E-cadherin in NPC was decreased but cytoplasmic expression was increased. The positive expression rate of membrane E-cadherin in NPI was significantly higher than that of NPC (P<0.01). Vimentin was localized in cytoplasm and negative in NPI epithelial cells, but the positive rate in NPC parenchymal cells was significant higher than that in NPI (P<0.01). The high expression of SATB1 in NPC was not related to the patents' sex, age, clinical classification and N classification, but positively correlated with T and M classification (P<0.05). Besides, high expression of SATB1 was positively correlated with vi-mentin in NPC tissues (r=0.358, P=0.009). SATB1 expression in NPC cell lines was negatively correlated with the levels of cell differentiation. Knockdown of SATB1 expression in C666-1 cells with siRNA was accompanied by an increase in E-cadherin and a decrease in vimentin levels, as well as a decrease in cell invasiveness. CONCLUSION:High expression of SATB1 promotes the clinical progress of NPC through EMT mechanism.     

Transforming growth factor-β1 regulates hypoxia-induced bronchial epithelial-mesenchymal transition by activation of lysys oxidase

AIM: To investigate whether transforming growth factor-β₁ (TGF-β₁) participates in hypoxia-induced bronchial epithelial-mesenchymal transition (EMT) through lysyl oxidase (LOX). METHODS: Sprague-Dawley (SD) rats were exposed to hypoxia to establish the animal model and were treated with LOX inhibitor β-aminopropionitrile (β-APN). Furthermore, primary rat bronchial epithelial cells were cultured in vitro and exposed either to normoxia or to hypoxia. TGF-β₁, TGF-β₁ receptor inhibitor (SB431542) or β-APN was used in the cell experiments. The content of collagen was measured by colorimetric method. The expression of TGF-β₁, LOX, and 2 EMT-related proteins (namely, the epithelial marker E-cadherin and the mesenchymal marker vimentin) were determined by immunohistochemistry and We-stern blot, respectively. RESULTS: The expression of TGF-β₁, vimentin and LOX and cross-linking of collagen were enhanced in hypoxia-exposed rat and in hypoxia-exposed bronchial epithelial cells, but the enhancement was impaired by the treatment with β-APN. In contrast, the expression of E-cadherin was reduced in hypoxia-exposed rat, and was reversed by treatment with β-APN. In vitro experiments demonstrated that TGF-β₁ and hypoxia led to the morphological phenotype characteristic of EMT in rat bronchial epithelial cells, in which the morphology of rat bronchial epithelial cells was switched from cobble-stone shape in normoxia-exposed group to spindle fibroblast-like morphology in hypoxia-or TGF-β₁-exposed group (P<0.01). Additionally, both β-APN and SB431542 partially prevented TGF-β₁ and hypoxia induced EMT in rat bronchial epithelial cells. TGF-β₁was able to dose-dependently up-regulate LOX expression in rat bronchial epithelial cells, which was blocked by concurrent incubation with SB431542. The up-regulation of TGF-β₁, vimentin, LOX and cross-linking of collagen and down-regulation of E-cadherin in hypoxia-exposed rat bronchial epithelial cells was significantly reversed by incubation with SB431542. CONCLUSION: TGF-β₁ regulates hypoxia-induced EMT in bronchial epithelial cells via activation of the LOX.     

Inhibitory effect of oxymatrine on high glucose-induced rat renal tubular epithelial-mesenchymal transition

AIM:To investigate the inhibitory effect of oxymatrine (OM) on high glucose-induced rat renal tubular epithelial-mesenchymal transition (EMT). METHODS:The rat renal tubular epithelial NRK52E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+different concentrations of OM groups and high glucose+0.50 g/L OM dynamic observation group. The expression of TGF-β1, Smad7, α-SMA and E-cadherin at mRNA and protein levels was detected by real-time PCR and Western blotting. The viability of NRK52E cells was determined by MTT assay. RESULTS:(1) Compared with control group, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose group gradually increased, and Smad7 protein and E-cadherin mRNA and protein gradually reduced, but the mRNA expression of Smad7 gradually increased. (2) Compared with high glucose group, as increases in OM doses, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose+different concentrations of OM groups gradually reduced, and Smad7 protein and E-cadherin mRNA and protein gradually increased, but the mRNA expression of Smad7 had no significant change. (3) Compared with high glucose group, the expression of TGF-β1 and α-SMA at mRNA and protein levels was significantly reduced, the expression of E-cadherin at mRNA and protein levels significantly increased, and the protein expression of Smad7 significantly increased, but the mRNA expression of Smad7 had no significant change in high glucose+0.50 g/L OM dynamic observation group. CONCLUSION: In NRK52E cells, oxymatrine inhibits high glucose induced EMT by down-regulating TGF-β1 and up-regulating Smad7, thus preventing the fibrosis effect of TGF-β1/Smads signaling.     

Effects of G6PD silencing by siRNA on expression of HIF-1α in human colon cancer cells

AIM: To study the effects of glucose-6-phosphate dehydrogenase (G6PD) silencing by small interference RNA(siRNA) on the levels of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and hypoxia-inducible factor 1α(HIF-1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells. The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD. LoVo cells were cultured in vitro under hypoxic condition. NADPH levels were determined.The mRNA and protein levels of HIF-1α were analyzed by RT-PCR and Western blotting,respectively. RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells. Compared with untransfected cells,the mRNA expression of G6PD in transfected cells was decreased by 43% and G6PD activity was decreased by 63.5%. Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased (41% vs 100%, P<0.05).HIF-1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells. CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF-1α stability in cancer cells under hypoxic condition. By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.     

Ligustilide inhibits angiogenesis and epithelial-mesenchymal transition in human hemangioendothelial cells by reducing VEGF levels

AIM To investigate the effect of ligustilide on human hemangioendothelial cells (HemECs) and to analyze its mechanism. METHODS The effect of ligustilide at different concentrations on the viability of HemECs was measured by CCK-8 assay. The HemECs were divided into control group and ligustilide （10, 25 and 50 μmol/L） treatment groups, and the proliferation of HemECs was detected by EdU staining. The effects of ligustilide on the angiogenesis of HemECs was tested by microtubule formation experiment. The protein expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition (EMT)-related markers in HemECs cells was determined by Western blot. RESULTS There was no significant difference in the viability of the cells treated with ligustilide at the concentrations between 0.1~50 μmol/L compared with control cells. Compared with control group, ligustilide at 25 and 50 μmol/L significantly reduced the number of EdU-positive cells and microtubule-like structures (P<0.05), reduced the protein expression level of VEGF (P<0.05), increased the protein expression of E-cadherin, and decreased the protein expression of vimentin and β-catenin (P<0.05). Compared with control group, the expression of VEGF and vimentin was significantly up-regulated, and the protein expression of E-cadherin was significantly down-regulated in VEGF overexpression group (P<0.05). Compared with VEGF overexpression group, the expression of VEGF and vimentin in 50 μmol/L ligustilide-treated VEGF-overexpressing cells were significantly reduced (P<0.05), and the protein expression of E-cadherin was significantly increased (P<0.05). CONCLUSION Ligulide inhibits the proliferation of HemECs, and also inhibits the angiogenesis and EMT process of HemECs by reducing the level of VEGF.     

Effects of TRPC1 on TGF-β1-induced epithelial-mesenchymal transition of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the epithelial-mesenchymal transition (EMT) of human bronchial epithelial (HBE) cells induced by transforming growth factor-β1 (TGF-β1). METHODS: EMT of 16HBE cells induced by TGF-β1 were identified by microscopy, immunofluorescence and Western blotting. Immunofluorescence, real-time PCR and Western blotting were applied to detect the mRNA and the protein expression of TRPC1 in the 16HBE cells. The influence of SKF96365 (a TRPC1 blocker) and siRNA-mediated silencing of TRPC1 on the EMT of the 16HBE cells were detected by microscopy and Western blotting. RESULTS: Treatment with TGF-β1 induced significant morphological changes of the 16HBE cells. Exposure to TGF-β1 decreased the expression of E-cadherin protein (P<0.01) and increased the expression of α-SMA protein (P<0.05) in the 16HBE cells. Immunofluorescence observation indicated that TRPC1 expression in the 16HBE cells was positive. The expression of TRPC1 at mRNA and protein levels was significantly increased in the 16HBE cells after stimulation with TGF-β1 (P<0.05). The morphological changes of the 16HBE cells induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group. The protein expression of E-cadherin and α-SMA induced by TGF-β1 were inhibited by SKF96365 and TRPC1 silencing compared with TGF-β1 group (P<0.05). CONCLUSION: TGF-β1 induces EMT with the mechanism of up-regulating TRPC1 in human bronchial epithelial cells.