A Study On The Role Of TGFβ1 Inducing HSCs To CAFs In Invasion And Metastasis Of SMMC-7721 Cell

Objective By using transwell co-culture model in vitro between HSCs(hepatic stellate cells)LX2 and human HCC(hepatocellular carcinoma)cell line SMMC-7721,the research aims to verify that HSCs will transformate into CAFs(cancer-associated fibroblast),which further enhances the EMT(epithelial-mesenchymal transition)process,invansive and metastasic capacity of HCC cell in the microenvironment.The results will contribute us to understand the role of the mesenchymal cells on invasion and metastasis of HCC better.Methods(1)Co-cultured LX2 cell and SMMC-7721 cell,observe the morphological changes of LX2 cell.TGF-beta1 treated LX2 cell,and observe the morphological changes of LX2 cell.(2)By different concentrations of TGF-beta1 and its inhibitor treating LX2 cell alone or co-cultured with SMMC-7721 cell,quantitative Real-Time PCR detected the mRNA expression level changes of CAFs related moleculars α-SMA,FAP,Desmin,FSP1.Western Blot detected protein expression level changes of α-SMA,FAP,Desmin.(3)By different concentrations of TGF-beta1 and its inhibitor treating SMMC-7721 cell alone or co-cultured with LX2 cell,Transwell migration assay and invasion assay detected the migrative and invasive ability changes of SMMC-7721 cell.(4)By different concentrations of TGF-beta1 and its inhibitor treating SMMC-7721 cell alone or co-cultured with LX2 cell,quantitative Real-Time PCR detected the mRNA expression level changes of SMMC-7721 cells with EMT related moleculars E-cadherin,N-Cadherin,N-Cadherin,Vimentin,Fibronectin,Twist,Snail.Western Blot detected protein expression level changes of E-Cadherin,Vimentin.(5)By different concentrations of TGF-beta1 and its inhibitor treating SMMC-7721 cell alone or co-cultured with LX2 cell,quantitative Real-Time PCR detected the mRNA expression level changes of SMMC-7721 cell with invasive migration related molecules EGF,VEGF,MMPs.Western Blot detected protein expression level changes of MMP2.Results(1)LX2 cell was co-cultured with SMMC-7721 cell for 48 h and was treated with TGF-beta 1 for 48 h,and the morphology of LX2 was transformed into a fibroblast cell like morphology.The morphology of a star or polygon turned into flat long spindle,cell synapse decreased significantly,and cells growed toward the poles;(2)When co-cultured with SMMC-7721 cell,mRNA expression level of α-SMA in LX2 cell increased significantly.Treated with different concentrations of TGF-beta1 and its inhibitor,TGF-beta1 can increase the expression of α-SMA and FAP significantly,and the inhibitor can inhibit the role of TGF-beta1.When co-cultured with SMMC-7721 cell,the protein expression level of α-SMA,Desmin was significantly increased.Treated with different concentrations of TGF-beta1 and its inhibitor,TGF-beta1 can increase the expression of α-SMA,Desmin,FAP significantly,and the inhibitor can inhibit the role of TGF-beta1.(3)When co-cultured with LX2 cell,the migrative and invasive SMMC-7721 cell number was increased significantly.Treated with different concentrations of TGF-beta1 and its inhibitor,TGF-beta1 can increase the migrative and invasive SMMC-7721 cell number significantly,and the inhibitor can inhibit the role of TGF-beta1.(4)When co-cultured with LX2 cell,the mRNA expression level of SMMC-7721 cell epithelial marker E-cadherin was decreased,the mRNA expression level of mesenchymal markers N-Cadherin,Vimentin was significantly increased,and that of the transcription factor of EMT regulation Twist was increased significantly.Treated with different concentrations of TGF-beta1 and its inhibitor,TGF-beta1 can decrease the expression of E-Cadherin,and increase the expression of N-Cadherin,Vimentin,Fibronectin,Snail significantly,and the inhibitor can inhibit the role of TGF-beta1.When co-cultured with LX2 cell,the protein expression level of E-cadherin was decreased,and that of Vimentin was significantly increased.Treated with different concentrations of TGF-beta1 and its inhibitor,TGF-beta1 can decrease the expression of E-Cadherin,and increase the expression of Vimentin significantly,and the inhibitor can inhibit the role of TGF-beta1.(5)When co-cultured with LX2 cell,the mRNA expression level of SMMC-7721 cell with invasive migration related molecules EGF,VEGF,MMP1,MMP2,MMP9,MMP13 was significantly increased.Treated with different concentrations of TGF-beta1 and its inhibitor,TGF-beta1 can increase the expression of MMP1,MMP2,MMP9,MMP13 and VEGF significantly,and the inhibitor can inhibit the role of TGF-beta1.When co-cultured with LX2 cell,the protein expression level of MMP2 was significantly increased.Treated with different concentrations of TGF-beta1 and its inhibitor,TGF-beta1 can increase the expression of MMP2 significantly,and the inhibitor can inhibit the role of TGF-beta1.Conclusion(1)In HCC microenvironment,HSCs can be converted to CAFs,and TGF beta1 can promote the transformation of HSCs.(2)When HSCs turned into CAFs,CAFs can promote the EMT process and the invasion and metastasis of HCC cell.(3)The results contribute us to understand the mesenchymal cells on invasion and metastasis of HCC better.