Role Of C-phycocyanin On Epithelial-mesenchymal Transformation Of Cervical Cancer Caski Cells Induced By TGF-?1

Objective: To investigate the effect of C-phycocyanin(C-PC)on epithelial-mesenhymal transformation(EMT)of cervical cancer cells induced by transforming growth factor-?1(TGF-?1).Methods: Cervical cancer Caski cells were induced by TGF-?1 of 10ng/m L.The morphological changes of cervical cancer cells in uninduced group and TGF-?1 induced group were observed under microscope.The expression levels of interstitial marker protein N-cadherin and epithelial marker protein E-cadherin were detected by western blot.Then the experiment was divided into three groups: control group,TGF-?1(10ng/m L)treatment group,TGF-?1(10ng/m L)combined with C-PC(300?g/m L)treatment group.After 24 hours and 48 hours of treatment,scratch test and Transwell assay were used to detect the effect of C-PC on the migration and invasion of Caski cells induced by TGF-?1.Western blot and immunofluorescence assay were used to detect the effect of C-PC on the expression of epithelial phenotypic marker protein E-cadherin and interstitial phenotypic marker protein N-cadherin in Caski cells induced by TGF-?1.Then,western blot was used to detect the effect of C-PC on TGF-?/ smads signal pathway in cervical cancer Caski cells induced by TGF-?1.Real-time quantitative PCR was used to detect the m RNA expression of interstitial phenotypic related transcription factors(Snail,Zeb1,Twist).In addition,flow cytometry was used to detect the effect of C-PC combined with TGF-?1 on the cell cycle of cervical cancer Caski cells for 24 h and 48 h,and the expression of cell cycle related protein(Cyclin D1,p21,p27)was detected by western blot.Results: 1.After TGF-?1 stimulation of cervical cancer Caski cells,obvious morphological changes were observed under microscope.The morphology of epithelial phenotypic cells with tight arrangement gradually changed to that of loosely arranged stromal phenotypic cells,and lost the original epithelial phenotypic characteristics.2.The results of scratch test and Transwell test showed that the migration and invasion ability of cells treated with TGF-?1was significantly stronger than that of the control group.TGF-?1 combinedwith C-PC treatment reversed the enhanced migration and invasion ability of cervical cancer Caski cells treated with TGF-?1 for 48 hours,suggesting that C-PC may inhibit the invasion and migration ability of cervical cancer Caski cells with EMT.3.Western blot showed that TGF-?1 alone down-regulated the expression of epithelial phenotypic marker protein E-cadherin,but had no significant effect on the protein expression of N-cadherin.TGF-?1 combined with C-PC treatment reversed the phenomenon of TGF-?1 group,upregulated the expression level of epithelial phenotypic marker protein E-cadherin,and down-regulated the protein expression level of interstitial phenotypic marker protein N-cadherin.4.Real-time quantitative PCR showed that compared with TGF-?1 alone,TGF-?1 combined with C-PC decreased the m RNA and protein expression levels of EMT-related transcription factors Twist,Snail and Zeb1,which confirmed that C-PC could inhibit the occurrence of epithelial-mesenchymal transformation.5.Flow cytometry showed that compared with the control group,TGF-?1 could reduce the number of cells in G0/G1 phase,increase the number of cells in G2/M phase and accelerate the progress of cell cycle.Compared with TGF-?1 group,TGF-?1 combined with C-PC could block the cell cycle in G0/G1 phase,and western blot suggested that C-PC might down-regulate Cyclin D1,up-regulation of p21 protein expression hinders the promoting effect of TGF-?1 on cell cycle progression.6.On this basis,Western blot found that the expression level of TGF-? type ?receptor in TGF-?1 treatment group was significantly higher than that in the control group,and had no significant effect on the protein level of psmad2/3,but increased the expression level of total protein smad2/3.The expression levels of TGF-? type ? receptor and psmad2/3 in TGF-?1 combined with C-PC treated cells were significantly inhibited.Conclusion: C-Phycocyanin treatment could inhibit the invasion and metastasis ability of cervical cancer Caski cells induced by TGF-?1,and inhibit the process of epithelial-mesenchymal transformation,which may be achieved by inhibiting TGF-?/smads signal pathway and blocking cell cycle progression.