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.     

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.     

Role of HMGA2 in epithelial-mesenchymal transition of gastric cancer cells

AIM:To investigate the role of HMGA2 in the epithelial-mesenchymal transition (EMT) in gastric cancer cells. METHODS:The expression of HMGA2 in human gastric cancer cell lines with different degrees of differen-tiation (MKN45, MKN28 and SGC7901) and immortalized human gastric epithelial cell line GES-1 was determined by Western blot and RT-qPCR. pcDNA3.0-HMGA2 plasmid was transfected into the MKN28 cells by liposome method. Transfection of si-HMGA2 interference fragments into MKN45 cells was also performed. The transfection efficiency was evaluated by Western blot and RT-qPCR. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the cell viability were measured by CCK-8 assay. The effects of HMGA2 over-expression in the MKN28 cells on the cell migration and invasion abilities were detected by wound healing and Transwell invasion assays. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the expression of EMT-related markers E-cadherin, N-cadherin, vimentin at mRNA and protein levels were determined by RT-qPCR and Western blot. The changes of Wnt/β-catenin signaling pathway-related molecules in the MKN28 cells with HMGA2 over-expression were also determined by RT-qPCR. RESULTS:The expression levels of HMGA2 were quite different in different differentiation levels of gastric cancer cells (P<0.05). The increased expression level of HMGA2 in MKN28 cells inhibited the cell viability (P<0.05), while the decreased expression level of HMGA2 in MKN45 cells promoted the cell viability (P<0.05). The increased expression level of HMGA2 in MKN28 cells promoted cell migration and invasion (P<0.05), changed the expression of EMT-related markers (P<0.05), while the decreased expression level of HMGA2 in the MKN45 cells changed the expression of EMT-related markers (P<0.05). The increased expression level of HMGA2 in the MKN28 cells significantly increased the mRNA levels of β-catenin in the Wnt/β-catenin pathway and the downstream molecules c-Myc and cyclin D1 (P<0.05). CONCLUSION:HMGA2 is closely related to the migration and invasion abilities of gastric cancer cells. Moreover, it promotes the EMT process of gastric cancer cells by activating Wnt/β-catenin pathway.     

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 c-SKI on coronary endothelial cell proliferation and endothelial-mesenchymal transition

AIM: To investigate the effect of cellular Sloan-Kettering Institute (c-SKI) on the proliferation and endothelial-mesenchymal transition of human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were treated with transforming growth factor-β1 (TGF-β1) at varying concentrations for different time points. Western blot was used to test the expression of c-SKI and mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin. Meanwhile, the endothelial marker E-cadherin was also detected. HCAECs were transfected with c-ski gene mediated by lentivirus (LV), the efficiency of LV-SKI transfection was detected by RT-qPCR. The HCAECs were divided into 4 groups:control group, TGF-β1 (5 μg/L) group, LV-SKI+ TGF-β1 group, LV-NC+ TGF-β1 group. The cell viability and colony formation were measured by MTT assay and colony formation assay. The protein levels of vimentin, α-SMA, E-cadherin, Smad2, Smad3, p-Smad2 and p-Smad3 were determined by Western blot. RESULTS: The expression of c-SKI was down-regulated in the HCAECs treated with TGF-β1 (P<0.01). Over-expression of c-SKI inhibited the proliferation of HCAECs (P<0.01). Compared with LV-NC group, over-expression of c-SKI down-regulated the expression of α-SMA and vimentin (P<0.01), up-regulated the expression of E-cadherin (P<0.01), and inhibited the protein phosphorylation of Smad2 and Smad3 (P<0.01), reversed the endothelial-mesenchymal transition induced by TGF-β1. CONCLUSION: The expression of c-SKI in the HCAECs is down-regulated in the process of endothelial-mesenchymal transition. Over-expression of c-SKI inhibits proliferation and endothelial-mesenchymal transition of HCAECs, the mechanism may be related to regulation of the TGF-β1/Smad signaling pathway.     

Calreticulin promotes migration and invasion of nasopharyngeal carcinoma cells by inducing cell EMT

AIM:To observe the expression of calreticulin (CRT) in nasopharyngeal carcinoma tissues, analyze the significance of clinical pathology and the influence on epithelial-mesencymal transition (EMT) of CNE2 cells. METHODS:The expression of calreticulin was detected by immunohistochemistry in 52 nasopharyngeal carcinoma and 57 nasopharyngeal benign tissues, and the significance of clinical pathology was evaluated. The calreticulin gene-specific small interfering RNA was constructed, and then was transfected into the NPC cell line CNE2 using the cationic liposome method. The effect of CRT on the morphological changes of the CNE2 cells was observed under light microscope. The effect of CRT on the cell migration and invasion abilities of the CNE2 cells was detected by Transwell migration and invasion assays. The expression of EMT-related proteins E-cadherin, vimentin, transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 in the CNE2 cells was determined by Western blot. RESULTS:The positive expression rate of CRT in the benign lesion tissues was 19.29% (11/57), which was significantly increased in the nasopharyngeal carcinoma tissues as 82.69% (43/52). The expression rate of CRT was positively correlated with the stage of nasopharyngeal carcinoma and lymph node metastasis (P<0.05). Knockdown of CRT expression made the CNE2 cells showing a spindle shape to a flat, cobblestone-like epithelial state change, arranged more compact, and the migration and invasion abilities were significantly decreased (P<0.05). Knockdown of CRT expression resulted in significant increase in the protein expression of E-cadhe-rin, and the decreases in the protein expression of vimentin, TGF-β and MMP-9 in the CNE2 cells (P<0.05). CONCLUSION:Calreticulin expression in nasopharyngeal carcinoma is significantly higher and positively correlated with nasopharyngeal carcinoma stage and lymph node metastasis. Calreticulin promotes cell migration and invasion of nasopharyngeal carcinoma CNE2 cells by inducing EMT.     

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.     

Reverse effect of shikonin on epithelial-mesenchymal transition induced by HGF in lung cancer cells

AIM:To investigate the effects of shikonin on the migration, invasion and epithelial-mesenchymal transition (EMT) in human non-small-cell lung cancer PC9 cells induced by hepatocyte growth factor (HGF). METHODS:The effect of shikonin on the viability of PC9 cells was measured by MTT assay. The cell migration and invasion abilities were analyzed by wound healing assay and Transwell method, respectively. The protein expression levels of E-cadherin, N-cadherin and vimentin in the PC9 cells were determined by Western blot. RESULTS:The viability of PC9 cells was significantly inhibited by shikonin in a dose-dependent manner (P<0.01), with IC₅₀ at 9.364 μmol/L. HGF significantly promoted the abilities of migration and invasion, and induced EMT in the PC9 cells. Shikonin significantly inhibited HGF-induced migration and invasion in the PC9 cells. The expression of E-cadherin was significantly down-regulated and the expression of N-cadherin and vimentin was significantly up-regulated in the presence of HGF (50 μg/L). However, shikonin reversed HGF-induced EMT, as indicated by up-regulation of E-cadherin and down-regulation of N-cadherin and vimentin (P<0.01). CONCLUSION:Shikonin reverses HGF-induced EMT in lung cancer PC9 cells.     

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.     

Over-expression of SCUBE2 suppresses epithelial-mesenchymal transition through Wnt/β-catenin signaling pathway in colorectal cancer cells

AIM: To study the effect of SCUBE2 on epithelial-mesenchymal transition (EMT) in colorectal cancer cells and its mechanism. METHODS: The expression of SCUBE2 in human colorectal cancer cell line HCT116 and normal colonic cell line FHC was detected by real-time PCR and Western blot. HCT116 cells were transfected with GV144-SCUBE2 to over-express SCUBE2, and then the cell viability, migration, and apoptosis were determined by MTT assay, Transwell assay and flow cytometry, respectively. The expression of EMT markers (E-cadherin, vimentin, and Snail), β-catenin, c-Myc and cyclin D1 in the HCT116 cells was analyzed by real-time PCR or Western blot after transfection with GV144-SCUBE2 for 6 h, followed by the stimulation of 10 μg/L recombinant TGF-β1 protein for 48 h. Additionally, the EMT process of HCT116 cells, which were stimulated by TGF-β1, over-expressed SCUBE2, and treated with Wnt/β-catenin pathway activator lithium chloride (LiCl) or inhibitor XAV93920, was analyzed by Western blot. RESULTS: Compared with FHC cells, the expression of SCUBE2 in the HCT116 cells was significantly decreased. The viability and migration ability of the HCT116 cells were suppressed by SCUBE2 over-expression, but the apoptosis was not markedly changed. Elevated expression of SCUBE2 increased E-cadherin expression, and decreased the expression of vimentin, Snail, β-catenin, c-Myc and cyclin D1 induced by TGF-β1. Treatment with LiCl blocked but treatment with XAV93920 enhanced the effects of SCUBE2 on EMT. CONCLUSION: Over-expression of SCUBE2 may inhibit the cell growth and migration, and suppress EMT through Wnt/β-catenin signaling pathway.     

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.     

miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells by repressing KLF10 expression

AIM:To explore the effect and the underlying mechanisms of microRNA-10b (miR-10b) on high glucose-stimulated epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. METHODS:The expression level of miR-10b was examined by RT-qPCR in the kidney tissues of the type 2 diabetes patients with kidney fibrosis. The EMT model of HK-2 cells was induced by high glucose stimulation and the miR-10b expression in the process was detected by RT-qPCR. The morphological changes of the HK-2 cells were observed using a microscope. EMT markers, such as fibronectin and N-cadherin, were examined by Western blot. The online database predicted that the 3'-UTR of KLF10 bound to miR-10b and their direct interaction was confirmed by dual luciferase report assay. RESULTS:Compared with the para-carcinoma normal tissues, the expression level of miR-10b was up-regulated in the tissues of type 2 diabetes patients with kidney fibrosis (P<0.01). In high glucose-stimulated HK-2 cells, the expression level of miR-10b was increased in a time-dependent manner (P<0.01). miR-10b inhibitor reversed the morphological changes and the increases expression of the EMT markers including fibronectin, SLUG, N-cadherin and SNAI1 induced by high glucose stimulation. Online database showed miR-10b was able to bind with the 3'-UTR in the promoter region of KLF10, thus negatively regulating its expression. Meanwhile, over-expression of KLF10 inhibited the EMT induced by high glucose. Inhibition of TGF-β/Smad3 activation was observed during the process of KLF10-repressed EMT. CONCLUSION:miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells may through repressing KLF10 expression.     

Muscarinic receptor 3 agonist promotes epithelial-meschymal transition in human lung cancer A549 cells by activating PI3K/AKT signaling pathway

AIM: To investigate the possible signaling pathway that promotes epithelial-mesenchymal transition (EMT) of the lung cancer A549 cells stimulated with muscarinic receptor 3 (M3R) agonist carbachol. METHODS: The lung cancer cells A549 were treated with 400 μmol/L carbachol. The morphological changes of the cells were observed under inverted phase contrast microscope. The migration and invasion abilites were measured by Wound healing and Transwell assays. qPCR was used to detect the mRNA level of vimentin and E-cadherin. The protein levels of p-AKT, vimentin and E-cadherin were determined by Western blot. RESULTS: After treatment with carbachol, the A549 cells showed loss of the close connection and the cell morphology was transformed from irregular polygon to spindle-like cells. The results of Wound healing and Transwell assays showed that the migration and invasion abilites of the A549 cells were enhanced. Carbachol increased the vimentin expression and decreased the E-cadherin expression at mRNA and protein level (P<0.05). The phosphorylation of AKT in the A549 cells was up-regulated (P<0.05). These changes was inhibited by M3R antagonist 4-DAMP. CONCLUSION: Carbachol promotes EMT in the human lung cancer A549 cells by activating PI3K/AKT signaling pathway.     

Effect of DEC1 gene silencing on viability,invasion and migration of human breast cancer MDA-MB-231 cells under hypoxia

AIM: To investigate the effect of differentiated embryonic chondrocyte gene 1 (DEC1) expression silencing on viability, invasion and migration of human breast cancer MDA-MB-231 cells and its possible mechanism under hypoxia. METHODS: The expression of DEC1 was detected by RT-qPCR and Western blot in breast cancer MDA-MB-231 cells under normoxia and hypoxia. MDA-MB-231 cells were transfected with the siRNA targeting DEC1 and the protein levels of DEC1, Smad3 and phosphorylated Smad3 (p-Smad3) were examined under hypoxia. Subsequently, the changes in the viability, invasion and migration abilities of MDA-MB-231 cells were analyzed by CCK-8 assay, Transwell experiment and Scratch test, respectively. RESULTS: The expression of DEC1 in MDA-MB-231 cells under hypoxia was higher than that in the MDA-MB-231 cells under normoxia condition at both mRNA and protein levels (P<0.05). The viability, invasion and migration abilities of MDA-MB-231 cells in siRNA-DEC1 group were decreased significantly as compared with control group (P<0.01). Besides, the protein level of p-Smad3 in the MDA-MB-231 cells in siRNA-DEC1 group was lower than that in negative control group under hypoxia condition (P<0.05). CONCLUSION: Down-regulated DEC1 expression significantly decreases the viability, invasion and migration abilities of breast cancer MDA-MB-231 cells by blocking the TGF-β/Smad3 signaling pathway under hypoxia condition.     

Inhibitory effect of metformin on alveolar epithelial-mesenchymal transition in lung tissue of rats with pulmonary fibrosis

AIM: To study the inhibitory effect of metformin on alveolar epithelial-mesenchymal transition (EMT) in rats with pulmonary fibrosis and the possible mechanism. METHODS: SD rats (n=48) were used, 12 of which were set up as normal control group, and 36 of which were induced by bleomycin (5 mg/kg) by tracheal instillation to establish pulmonary fibrosis. The pulmonary fibrosis rats were randomly divided into bleomycin group, low dose (100 mg/kg) of metformin group, and high dose (300 mg/kg) of metformin group. The rats in metformin groups were given the corresponding dose of metformin daily for 4 weeks. HE staining and Masson staining were used to observe the changes of lung histopathology and collagen deposition. Real-time PCR, Western blot and innunohistochemical staining were used to detect the mRNA and protein expression of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, zonula occludens-1 (ZO-1), collagen I, collagen III and transforming growth factor-β₁ (TGF-β₁), and the protein phosphorylation levels of Smad2/3 and extracellular signal-regulated kinase 1/2 (ERK1/2) were also determined. RESULTS: Metformin up-regulated the expression of E-cadherin and ZO-1, down-regulated the expression of α-SMA, vimentin, collagen I and collagen III, and the protein phosphorylation levels of Smad2/3 and ERK1/2 were also decreased (P<0.05). CONCLUSION: Metformin inhibits alveolar EMT in the rats with pulmonary fibrosis, and its mechanism may be related to the inhibition of TGF-β₁ signal transduction pathway.     

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

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

miR-125a-5p suppresses epithelial-mesenchymal transition via GSK-3β/Snail signaling pathway in breast cancer cells

AIM: To investigate the function of microRNA-125a-5p (miR-125a-5p) on epithelial-mesenchymal transition (EMT) of breast cancer cells via GSK-3β/Snail signaling pathway.METHODS: The expression of miR-125a-5p in normal breast epithelial cells and breast cancer cells, as well as the transfection efficiency of miR-125a-5p plasmid in MDA-MB-231 cells was detected by RT-qPCR. The chemotaxis ability and invasion ability were detected by chemotaxis assay and Transwell invasion assay. The changes of EMT-related markers, the protein level of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) and the nuclear translocation of Snail were determined by Western blot. RESULTS: The expression of miR-125a-5p in the breast cancer cells was significantly lower than that in the normal breast epithelial cells. The expression of miR-125a-5p was significantly higher in MDA-MB-231/miR-125a-5p cells than that in MDA-MB-231/NC cells. The ability of epithelial growth factor (EGF) at 10 μg/L to induce chemotaxis of MDA-MB-231 cells was the strongest. Compared with MDA-MB-231/NC group, stimulation of EGF decreased the invasion ability of MDA-MB-231/miR-125a-5p cells, and resulted in the increase in E-cadherin expression, while significantly decreased the protein levels of vimentin and p-GSK-3β. Meanwhile, the nuclear localization of Snail was significantly inhibited. The invasion capacity of MDA-MB-231/miR-125a-5p+GAB2 cells was significantly enhanced compared with MDA-MB-231/miR-125a-5p+Con cells, the expression of E-cadherin was decreased, and the protein levels of vimentin and p-GSK-3β were significantly increased, while the nuclear localization of Snail was promoted. CONCLUSION: miR-125a-5p suppresses EMT via GSK-3β/Snail signaling pathway, thus inhibiting the invasion ability of breast cancer cells.     

Effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45

AIM: To investigate the effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45. METHODS: The protein levels of CADM1 in 3 human gastric carcinoma cell lines were detected by Western blotting. Eukaryotic expression vector pcDNA-CADM1 was constructed and transfected into MKN-45 cells. The MKN-45 cells stably expressing CADM1 were selected by G418 and identified by Western blotting. Furthermore, CCK-8 assay and Boyden chamber were used to analyze the effects of CADM1 overexpression on the prolife ration and invasion of gastric carcinoma cells. Western blotting was also utilized to detect the levels of cell proliferation- and invasion-related proteins. RESULTS: Relative level of CADM1 protein in MKN-45 cells was significantly lower than that in MKN-28 cells and SGC-7901 cells. Additionally, eukaryotic expression vector pcDNA-CADM1 was successfully constructed and MKN-45 cells stably expressing CADM1 were obtained. Compared with non-treatment and pcDNA3.1 groups, the proliferation of MKN-45 cells was obviously inhibited in pcDNA-CADM1 group. The result of Boyden chamber showed that the migrated cell numbers in pcDNA-CADM1 group (52.35±3.89) were significantly lower than that in untreated group (101.53±6.89) and pcDNA3.1 group (98.77±7.03). Compared with non-treatment and pcDNA3.1 groups, the protein level of p21 was significantly up-regulated and protein expression of MMP-2 and MMP-9 was obviously down-regulated. CONCLUSION: Overexpression of CADM1 may markedly inhibit cell proliferation and reduce invasion ability, and thus may be a novel target for treating gastric carcinoma.