Silybinin Inhibits Migration And Invasion Of Hepatocellular Carcinoma HepG2 Cells And The Related Mechanism

Objective:The goal of this study was to observe whether silybinin can inhibit the metastasis of HepG2 hepatocellular carcinoma(HCC)cell lines.In addition,N-cadherin is an important marker of epithelial-mesenchymal transition(EMT)in malignant tumor cells.We established the Epidermal growth factor(EGF)induced HepG2 cells EMT model in vitro to identify whether silibinin can reverse HepG2 cell EMT by decreasing expression of N-cadherin proteins.Furthemore,we also expored the molecular mechanisms.Methods:(1)Role study:MTT assay was used to determine the effect of silybinin on proliferation of HepG2 cells.The effect of silibinin on the migration of HepG2 cells was evaluated by cell scratch experiment.Migration and invasive capacity was evaluted by transwell assay for the HepG2 cells.mRNA transcription level of Twist,Snail,Slug,vascular endothelial growth factor(VEGF),MMP-11 and MMP-13 genes was qualified by real-time quantitative PCR.The proliferation of HepG2 cells,migration distance,the numbers of migrated cells and gene expression levels of Snail,Slug,VEGF,MMP-11 and MMP-13 were compared among four groups by One-way ANOVA.P<0.05 was considered statistically significant.(2)Mechanism study:We used different concentrations(0,1 0,20ng/mL)of EGF to stimulate HCC HepG2 cell.Cell morphological changes of HepG2 cells were observe by inverted microscope.N-cadherin,E-cadherin and Vimentin protein expression levels were detected by Western Blot.The cell migration and invasive capacity was observed by cell scratch and invasion assay,respectively.mRNA transcription levels of N-cadherin,E-cadherin and Vimentin gene were detected by real-time quanti-tative PCR and N-cadherin,E-cadherin and Vimentin protein expressions of HepG2 cells were detected by Western Blot.At the end of the experiment,we used Western Blot to detect the expression of ERK,Akt and MMP-9.Result:(1)Role study:MTT assay showed that silybinin(0,30,60,120,240 and 480?g/ml)treatment could significantly decrease viability of HepG2 cells in dose-and time-dependent manners(p<0.05).The results of scratch and transwell assays confirmed that silybinin treatment reduced the ability of migration and invasion of HepG2 cells in a dose-dependent manner.Real-time quantitative PCR results indicated that the expression of Twist,Snail and Slug was prominently inhibited in HepG2 cells(p<0.05),and silibinin can down-regulate the expression of VEGF and MMP-13 in different degrees,while had no significant effect on MMP-11 gene expression.(2)Mechanism study:After EGF treatment,morphology was converted to a diffused fibroblast-like morphology.The results of cell scratch and transwell assay showed that EGF could promote the migration and invasion of HCC HepG2 cells.Real-time quan titative PCR and Western Blot results demonstrated that EGF could significantly up-regulate Vimentin and N-cadherin expression,compared with the control group(p<0.05).However,EGF did not significantly down-regulate the expression of E-cadherin protein.Also it showed that silibinin could significantly decrease the expression of N-cadherin both on gene and protein level,but had no significant effect on the expression of Vimentin.In addition,Western Blot results indicated that silybinin significantly down-regulated the expression of ERK and Akt(p<0.05),while had no significant effect on MMP-9 expression.Conclusion:(1)Silybinin can inhibit the proliferation,migration and invasion of HCC HepG2 cells.(2)Silybinin treatment significantly down-regulae the expression of Twist,Snail,Slug,VEGF and MMP-13 genes.(3)In the EGF-induced HCC HepG2 cells EMT model,Silibinin can inhibit the expression of N-cadherin,reverse the EMT,and effectively inhibit HCC HepG2 cells migration and invasion.(4)Silibinin may inhibit HCC HepG2 migration and invasion through ERK1/2 and Akt pathway.