TGF-β Combined With EGFR Inhibitor Induce The Differentiation Of Hepatoma Cells By Influence EMT

Objective:Hepatocytes show multi-differentiation potential,which can be re-differentiated into normal hepatocytes in the case of injury.However,when microenvironment changes on account of incomplete healing inflammation,hepatocytes which could transform into fibroblasts participate in liver fibrosis.Our previous studies also found that hepatoma carcinoma cells could exhibit benign fibroblast characteristics after changing inflammatory tumor microenvironments.Recently,a study has shown that EMT derived breast cancer cells could be induced to trans-differentiate into post-mitotic and functional adipocytes.EMT is an inevitable intermediate process during cell differentiation and remodeling.The high plasticity of cells after EMT is the functional basis of cell trans-differentiation.The microenvironment,especially the growth factor level,has a significant impact on the EMT process,TGF-β is an important growth factor in cell differentiation.In our previous study,the induced HCC cells down-regulated expressed EGFR.Based on results of the pre-experiment,we found that TGF-β combined with EGFR inhibitor could induce HCC cells to show fibroblast characteristics significantly.Therefore,this study aimed to investigate the effect of TGF-β combined with EGFR inhibitor on the differentiation of hepatoma cells and its mechanism.Methods1.By setting the control group,TGF-β group,EGFR inhibitor group and TGF-βcombined with EGFR inhibitor group to culture hepatoma carcinoma cells.The induction effect of each treatment group on cells was observed.2.Flow cytometry was used to detect the expression of fibroblast surface markers CD34,CD11 b and CD105;immunofluorescence and western blot were used to detect the expression of fibroblast-associated protein;by constructing a nude mouse xenograft model,immunohistochemical staining was used to detect the expression of fiber-related proteins COL and FN in each group,masson staining was used to detect the expression of collagen in each group.3.Trypan blue count and CCK8 assay detected the effect of each treatment group on the proliferation;wound-healing experiment was carried out to examine the effects of treatment groups on migration ability;transwell assay was used to test the effect of each treatment group on the invasion ability.By constructing a nude mouse xenograft model,proliferation of liver cancer xenografts was examined.4.Flow cytometry was used to detect the expression of tumor stem-potential surface markers,tumor EMT-related markers in each group.Western blot was used to detect the expression of EMT-related proteins in each group.5.RNA sequence to analyze differential expression genes in each group;pathway enrichment analysis was performed for each group.6.By weighted gene co-expression network analysis and Protein Interaction Network for differential expression genes,the key molecules in which TGF-βcombined with EGFR inhibitors to induce hepatoma cells to differentiate were explored.7.The key molecule was verified by experiments.After inhibiting the key molecule,the cell morphology of the induced cells was observed;the expression of the surface of fibroblasts markers was detected by flow cytometry;western blot was used to detect the expression of fibroblast-associated protein.Results:1.TGF-β combined with EGFR inhibitor induces HCC cells to show characters of fibroblasts.HCC cells exhibited long spindle-like changes similar to fibroblast,highly expressed fibroblast surface markers CD34,CD11 and CD105 as well as fibroblast-associated protein Col I,FN,vim and α-SMA.2.TGF-β combined with EGFR inhibitor group significantly inhibited the proliferation,migration and invasion of HCC cells compared with other groups.3.In vivo experiments showed that TGF-β combined with EGFR inhibitor could significantly inhibit the growth of hepatoma cell xenografts compared with other treatment groups.Immunohistochemistry results showed that the combined group could induce HCC cells to highly express fibroblast-associated proteins in vivo.Masson staining showed that cells highly express collagen in vivo.4.TGF-β induced III EMT occurrence in HCC cell,cells highly expressed tumor stem-potential surface markers and tumor EMT-related markers,III EMT marker genes were up-regulated.TGF-β combined with EGFR inhibitor could not induce III EMT in HCC cell,no significant differential expression in tumor stem-potential surface markers and tumor EMT-related markers,and III EMT marker genes were down-regulated.5.Principal component analysis indicated that cells induced by TGF-β combined with EGFR inhibitor were different from the cells in control group and TGF-β group.II EMT marker genes and SAMes genes were highly expressed.The differentiation function was highly active in the combination group,and multiple fibroblast-related gene sets and the differentiation function gene sets of embryonic stem cells were significantly enriched.6.RUNX2 is a key gene for the differentiation of HCC cells induced by TGF-βcombined with EGFR inhibitors.RUNX2 is significantly associated with the phenotype induced by the combination group,and has significant interactions with fibroblast-related genes and mesenchymal marker genes.7.After the inhibition of RUNX2,HCC cells induced by TGF-β combined with EGFR inhibitors did not show fibroblasts characters.For RUNX2 related pathway,TGF-β/Smad3/ RUNX2 promoted the expression of fibrosis-related genes,while the ERK pathway activated by Runx2 was blocked by EGFR inhibitor,thereby inhibiting the malignancy of HCC.ConclusionTGF-β combined with EGFR inhibitors can induce HCC cells differentiation into fibroblasts,cell shows the characteristics of fibroblasts,and cell proliferation,migration and invasion ability are inhibited.TGF-β combined with EGFR inhibitors has affected the EMT process of HCC cells,cells underwent II EMT to fibroblasts differentiation.RUNX2 is a key gene for the differentiation,inhibit RUNX2 after the can reversible differentiation induced by the combination.