TGFβ1 Induces Transformation Of HSCs Into CAFs To Promote Cell Invasion And Metastasis Of HCC

ObjectiveIn this study,three HCC cell lines were co-cultured with LX2 cells by transwell to demonstrate that TGFβ1 in the tumor microenvironment could promote transformation of LX2 cells into CAFs.The transformed LX2 cells could then induce EMT process in HCC cell lines,and eventually contribute to cell migration and invasion in HCC.Methods① LX2 cells were co-cultured with HCC cell lines(including SMMC-7721,BEL-7402 and HepG2)or stimulated with TGFβ1 for 48 h,and then observe the cell morphological changes.② LX2 cells were co-cultured with HCC cell lines or treated with different concentrations of TGFβ1 and its inhibitor for 48 h,and then detect the expression of CAFs associated molecules including α-SMA,FAP and Desmin by quantitative Real-Time PCR and Western Blot.③ BEL-7402 cells(cultured alone or co-cultured with LX2 cells)were treated with different concentrations of TGFβ1 and its inhibitor for 48 h,and then detect the migration and invasion ability of the cells.④ HCC cell lines(cultured alone or co-cultured with LX2 cells)were treated with different concentrations of TGFβ1 and its inhibitor for 48 h,and then detect the expression of EMT-related molecules of HCC cells by quantitative Real-Time PCR and Western Blot.⑤ HCC cell lines(cultured alone or co-cultured with LX2 cells)were treated with different concentrations of TGFβ1 and its inhibitor for 48 h,and then detect the migration and invasion correlated moleculars by quantitative Real-Time PCR and Western Blot.Results① LX2 cells were co-cultured with HCC cells(SMMC-7721,BEL-7402,HepG2),or stimulated with TGFβ1 for 48 h.The morphological changes of LX2 cells were then observed.The cells turned from the star or polygon into flat long spindle shape.The cells showed polarized growth feature,and their rich cell synapses significantly reduced,bringing out the morphological features of myofibroblastst.② LX2 cells were co-cultured with HCC cells(SMMC-7721,BEL-7402,HepG2)for 48 h.The results of Real-Time PCR showed that the expression of CAFs related genes such as α-SMA and FAP were significantly up-regulated.LX2 cells(cultured alone or co-cultured with HCC cells)were treated with different concentrations of TGFβ1 and its inhibitor for 48 h.Results showed that the gene expression of α-SMA and FAP were significantly up-regulated by TGFβ1,and the effect of TGFβ1 could be inhibited by the inhibitor SB525334.③ BEL-7402 cells were co-cultured with LX2 cells for 48 h.Results showed that the migration and invasion abilitities of the BEL-7402 cells were significantly promoted after cocultured with LX2 cells.BEL-7402 cells(cultured alone or co-cultured with LX2 cells)were treated with different concentrations of TGFβ1 and its inhibitor for 48 h.The results of the experiment showed that TGFβ1 significantly enhanced the migration and invasion abilities of BEL-7402 cells,furthermore the effect in the co-cultured group was more remarkerable.The function of TGFβ1 could be suppressed by the inhibitor.④ HCC cell lines were co-cultured with LX2 cells for 48 h.The results of Real-Time PCR showed that the gene expression of E-cadherin in SMMC-7721 cells significantly decreased.Meanwhile,the gene expression of N-cadherin,Twist and Vimentin were significantly up-regulated.And the expression of fibronectin in BEL-7402 and HepG2 cells was significantly upregulated.At protein level,in three HCC cell lines(SMMC-7721,BEL-7402 and HepG2),E-cadherin protein siginificantly decreaed and vimentin significantly increased.HCC cell lines were treated with different concentrations of TGFβ1 and its inhibitor for 48 h.In SMMC-7721 cells,the gene expression of E-Cadherin was down-regulated,and vimentin was up-regulated under the effect TGFβ1 at mRNA level.The inhibitor could inhibit the effect of TGFβ1.The results of Western Blot showed that in HCC cells(SMMC-7721,BEL-7402 and HepG2)E-cadherin protein decreased and Vimentin protein significantly increased after being treated by TGFβ1.The inhibitor could inhibit the effect of TGFβ1.⑤ HCC cell lines were co-cultured with LX2 cells for 48 h.The results of Real-Time PCR showed that the expression of EGF,VEGF,MMP1,MMP2,MMP9 and MMP13 were significantly up-regulated in SMMC-7721 cells after being co-cultured with LX2 cells.Moreover,in BEL-7402 cells the gene expression of MMP1,MMP2 and MMP9 were significantly up-regulated;in HepG2 cells the gene expression of VEGF,MMP1 were significantly up-regulated after being co-cultured with LX2 cells.The results of Western Blot showed that after being cocultured with LX2 cells,the protein of MMP2 and VEGF significantly increased in HCC cells.HCC cell lines were treated with different concentrations of TGFβ1 and its inhibitor.The results of Real-Time PCR showed that the genes enpression of MMP2 and VEGF in SMMC-7721 cells was significantly up-regulated by TGFβ1.The effect of TGFβ1 could be inhibited by its inhibitor.The results of Western Blot showed that the protein of MMP2 in SMMC-7721 siginificantly increased under the influence of TGFβ1.Besides,the protein of MMP2 and VEGF in BEL-7402 and HepG2 cells significantly increased after being treated by TGFβ1.Inhibitor could inhibit the effect of TGFβ1.Conclusion① In HCC microenvironment,HSCs can transform into CAFs,and TGFβ1 promote the transformation process.② The transformation of HSCs into CAFs can promote the EMT process of HCC cells,and eventually enhance the migration and invasion abilities of HCC.③ These results provide experimental basis for the role of stromal cells in the invasion and metastasis of hepatocellular carcinoma.