Objective Epithelial mesenchymal transition(EMT)plays a very important role in ovarian cancer metastasis,and IL-8 released from mechanosensitive cancer cells may contribute to the EMT process of solid carcinomas.In th...Objective Epithelial mesenchymal transition(EMT)plays a very important role in ovarian cancer metastasis,and IL-8 released from mechanosensitive cancer cells may contribute to the EMT process of solid carcinomas.In this study,we have explored IL-8 and its receptors signal transduction process of human ovarian cancer cells under conditions of FSS,and simultaneously detected the EMT process of ovarian cancer.Methods After the fluid shear stress was loaded,LightCyclerTM system and ELISA were employed to assay the IL-8 mRNA expression and protein production,respectively.Meanwhile,IL-8 reporter gene pEGFP1-IL8USCS was constructed for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis.RT-PCR,Northern blot and immunofluorescence were used to determine the expression of IL-8 receptor CXCR2 at mRNA and protein levels.IL-8 downstream signaling molecule NF-κB nuclear translocation was observed by immunocytofluoresent staining.Western blot was used to examine IκB phosphorylation and EMT-related protein.Results(1)The increase of IL-8 mRNA expression by shear stress was time-dependent.The expression increased when SKOV3 cells exposed to fluid shear stress for 1 h,reached the summits at 2 h,gradually decreased at 3 h and remained at a constant level at 4~12 h.Additionally,IL-8 expression was negatively associated with the intensity of shear stress.After SKOV3 cells were exposed to low fluid shear stress(1.5 dyne/cm2)for 1 h and 2 h,IL-8 mRNA expression increased near 68 and 52 times respectively as that of SKOV3 cells exposed to a high fluid shear stress of 5.0 dyne/cm^2.(2)The productions of IL-8 protein in SKOV3 cells subjected to shear stress were time-dependent.The secretion reached the summit when SKOV3 cells exposed to fluid shear stress for 5 h,then IL-8 secretion gradually decreased at 8 h of stimulation by shear stress.IL-8 secretion increased obviously when fluid shear stress(0.5,1.5,or 2.0 dyne/cm2)was exerted on SKOV3 cells for 1 h.Notablely,the secretion of IL-8 was the highest when SKOV3 cells subjected to fluid shear stress 1.5dyne/cm^2,which was near 6 or 7 times as that of SKOV3 cells subjected to high fluid shear stress(5.0 dyne/cm^2).(3)There was an increase in enhanced green fluorescent protein expression in pEGFPI-IL8USCS-transfected SKOV3 cells subjected to a fluid shear stress of 1.5 dyne/cm2 for 2 h,suggesting a flow shear stress induced IL-8 gene transcriptional activation;(4)CXCR2,which was constitutively present on the surface of SKOV3 cells,increased following exposure to fluid shear stress for 60 min.(5)Following the application of a shear stress of 1.5 dyne/cm^2,NF-κB p65 became detectable in the cell nuclei and Phosphorylated IκB in cell lysates increased significantly;(6)Compared with the control group,critical EMT-related proteins vimentin was upregulated,E-cadherin was downregulated after the application of the 1.5 dyne/cm2shear stress for 2 h,which suggested the EMT of ovarian cancer.Conclusions FSS triggered IL-8/CXCR2 signaling of SK-OV3 cells represents an early gene activation and the activation can be mediated through NF-κB.When the fluid shear stress-induced IL-8/CXCR signaling activated,the expression of EMT-related proteins changed.This observation suggested that fluid shear stress-induced IL-8 activation and the downstream signal pathways may have important contribution to the EMT process of ovarian cancer cells.展开更多
OBJECTIVE Currently discussing the clinical treatment of pulmonary fibrosis commonly used drugs astragalus main active ingredient astragalosideⅣ(ASTⅣ) in vitro after transforming growth factor-β1 induced lung adeno...OBJECTIVE Currently discussing the clinical treatment of pulmonary fibrosis commonly used drugs astragalus main active ingredient astragalosideⅣ(ASTⅣ) in vitro after transforming growth factor-β1 induced lung adenocarcinoma A549 epithelial cells after epithelial cell interstitial EpithelialMesenchymal Transition(EMT).METHODS The effect of astragalosideIV on the proliferation of A549 cells was detected by MTT assay for the first time.No significant effect of astragaloside on the prolifera.tion of A549 cells was found in the range of 1.25-20 μmol/L.A549 cells in vitro were divided into 5 groups:normal group,control group,low,medium and high experimental groups,which were treated for 72 hours,and the morphological changes of cells in each group were observed by light microscope.Real-time quantitative PCR(qPCR) and Western blotting were performed.Detection of gene and protein expression levels.RESULTS The results of real-time fluorescence quantitative PCR showed that the quantitative analysis of high-dose astragalosideⅣ in the experimental group was lower than that of the control group in the α-SMA analysis,and the difference was statistically significant(P<0.05).The dose of Astragaloside Ⅳ in the experimental group was higher than that of the control group in the E-Cad analysis,and the difference was statistically significant(P<0.05).Western Blot results showed that the expression of α-SMA antibody in the high-dose and low-dose experimental group was lower than that in the control group,the difference was statistically significant(P<0.05).The high-dose experimental group had a significantly higher expression of E-Cad antibody than the control group,the difference was statistically significant(P<0.01).CONCLUSION This study uses A549 epithelial cells as a model,A549 cells were modeled and confirmed that Astragaloside can effectively inhibit TGF-β1-induced epithelialmesenchymal transition(EMT) and provide a new basis for the treatment of pulmonary fibrosis.展开更多
基金supported by Foundation of Sichuan Provincial Science and Technology Program ( 2019YFH0147,2019YFH0158)1. 3. 5 Project for Disciplines of Excellence,West China Hospital,Sichuan University ( ZYJC18016)
文摘Objective Epithelial mesenchymal transition(EMT)plays a very important role in ovarian cancer metastasis,and IL-8 released from mechanosensitive cancer cells may contribute to the EMT process of solid carcinomas.In this study,we have explored IL-8 and its receptors signal transduction process of human ovarian cancer cells under conditions of FSS,and simultaneously detected the EMT process of ovarian cancer.Methods After the fluid shear stress was loaded,LightCyclerTM system and ELISA were employed to assay the IL-8 mRNA expression and protein production,respectively.Meanwhile,IL-8 reporter gene pEGFP1-IL8USCS was constructed for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis.RT-PCR,Northern blot and immunofluorescence were used to determine the expression of IL-8 receptor CXCR2 at mRNA and protein levels.IL-8 downstream signaling molecule NF-κB nuclear translocation was observed by immunocytofluoresent staining.Western blot was used to examine IκB phosphorylation and EMT-related protein.Results(1)The increase of IL-8 mRNA expression by shear stress was time-dependent.The expression increased when SKOV3 cells exposed to fluid shear stress for 1 h,reached the summits at 2 h,gradually decreased at 3 h and remained at a constant level at 4~12 h.Additionally,IL-8 expression was negatively associated with the intensity of shear stress.After SKOV3 cells were exposed to low fluid shear stress(1.5 dyne/cm2)for 1 h and 2 h,IL-8 mRNA expression increased near 68 and 52 times respectively as that of SKOV3 cells exposed to a high fluid shear stress of 5.0 dyne/cm^2.(2)The productions of IL-8 protein in SKOV3 cells subjected to shear stress were time-dependent.The secretion reached the summit when SKOV3 cells exposed to fluid shear stress for 5 h,then IL-8 secretion gradually decreased at 8 h of stimulation by shear stress.IL-8 secretion increased obviously when fluid shear stress(0.5,1.5,or 2.0 dyne/cm2)was exerted on SKOV3 cells for 1 h.Notablely,the secretion of IL-8 was the highest when SKOV3 cells subjected to fluid shear stress 1.5dyne/cm^2,which was near 6 or 7 times as that of SKOV3 cells subjected to high fluid shear stress(5.0 dyne/cm^2).(3)There was an increase in enhanced green fluorescent protein expression in pEGFPI-IL8USCS-transfected SKOV3 cells subjected to a fluid shear stress of 1.5 dyne/cm2 for 2 h,suggesting a flow shear stress induced IL-8 gene transcriptional activation;(4)CXCR2,which was constitutively present on the surface of SKOV3 cells,increased following exposure to fluid shear stress for 60 min.(5)Following the application of a shear stress of 1.5 dyne/cm^2,NF-κB p65 became detectable in the cell nuclei and Phosphorylated IκB in cell lysates increased significantly;(6)Compared with the control group,critical EMT-related proteins vimentin was upregulated,E-cadherin was downregulated after the application of the 1.5 dyne/cm2shear stress for 2 h,which suggested the EMT of ovarian cancer.Conclusions FSS triggered IL-8/CXCR2 signaling of SK-OV3 cells represents an early gene activation and the activation can be mediated through NF-κB.When the fluid shear stress-induced IL-8/CXCR signaling activated,the expression of EMT-related proteins changed.This observation suggested that fluid shear stress-induced IL-8 activation and the downstream signal pathways may have important contribution to the EMT process of ovarian cancer cells.
基金supported by Grants from the National Natural Science Foundation of China (81373795 81473526)
文摘OBJECTIVE Currently discussing the clinical treatment of pulmonary fibrosis commonly used drugs astragalus main active ingredient astragalosideⅣ(ASTⅣ) in vitro after transforming growth factor-β1 induced lung adenocarcinoma A549 epithelial cells after epithelial cell interstitial EpithelialMesenchymal Transition(EMT).METHODS The effect of astragalosideIV on the proliferation of A549 cells was detected by MTT assay for the first time.No significant effect of astragaloside on the prolifera.tion of A549 cells was found in the range of 1.25-20 μmol/L.A549 cells in vitro were divided into 5 groups:normal group,control group,low,medium and high experimental groups,which were treated for 72 hours,and the morphological changes of cells in each group were observed by light microscope.Real-time quantitative PCR(qPCR) and Western blotting were performed.Detection of gene and protein expression levels.RESULTS The results of real-time fluorescence quantitative PCR showed that the quantitative analysis of high-dose astragalosideⅣ in the experimental group was lower than that of the control group in the α-SMA analysis,and the difference was statistically significant(P<0.05).The dose of Astragaloside Ⅳ in the experimental group was higher than that of the control group in the E-Cad analysis,and the difference was statistically significant(P<0.05).Western Blot results showed that the expression of α-SMA antibody in the high-dose and low-dose experimental group was lower than that in the control group,the difference was statistically significant(P<0.05).The high-dose experimental group had a significantly higher expression of E-Cad antibody than the control group,the difference was statistically significant(P<0.01).CONCLUSION This study uses A549 epithelial cells as a model,A549 cells were modeled and confirmed that Astragaloside can effectively inhibit TGF-β1-induced epithelialmesenchymal transition(EMT) and provide a new basis for the treatment of pulmonary fibrosis.
