IER2 Regulates The Migration And Invasion Of Liver Cancer Cells Through The ITGB1-mediated Signaling Pathway

Objective:The leading cause of death for the vast majority of hepatocellular carcinoma patients is a result of the direct metastases,which is a complex process involved abnormal expression of multiple genes and associated signal pathways.Human immediate early response 2(IER2)has been characterized as a putative nuclear protein that served function as an early transcription factor or transcriptional co-activator in the regulation of cellular responses.Currently,evidence suggests that IER2 seems to play a positive role in the migration and invasion of pancreatic cancer cells and breast cancer cells,however,little is known about its biological role and the mechanism involved.In the present study,we aimed to investigate the effect of IER2 on HCC cell motility and cell-extracellular matrix(ECM)adhesion and spreading,and to unveil the underlying mechanisms by which IER2 regulated HCC cell motility and adhesion。Methods:The HCC cell lines were selected,and the recombinant lentiviruses encoding IER2,shRNA against human IER2,and indicated control lentiviruses were generated or acquired.After lentiviral-infection,cells were screened by using puromycin to select stably transduced cell lines and were performed to investigate the effects of IER2 on cell migration,invasion and cell-ECM adhesion.(1)RT-qPCR and Western blot analyses were used to evaluate the expression of IER2 in four HCC cell lines with different metastatic potential.(2)Cell Counting Kit-8 assay was performed to study the effect of overexpression and knockdown of IER2 on the viability of SMMC-7721 and MHCC97H cells.(3)Transwell cell migration and invasion assays were performed to assess the effects of overexpression and knockdown of IER2 on migration and invasion of SMMC-7721 and MHCC97H cells.(4)HCC cell-ECM adhesion and spreading assays was performed to investigate the effects of overexpression and knockdown of IER2 on adhesion onto different matrix and spreading on Fibronectin of the SMMC-7721 and MHCC97H cells.(5)The effects of overexpression and knockdown of IER2 in SMMC-7721 cells and in MHCC97H cells on ITGA5 or ITGB1 expression both in mRNA and protein levels were confirmed by RT-qPCR and western blot analysis.Meanwhile,Luciferase reporter gene assay was used to verify whether the observed positive correlation of IER2 with the ITGB1 expression is the consequence of the transcriptional regulation of ITGB1.Moreover,Chromatin immunoprecipitation assay was further performed to clarify whether IER2 may interact with ITGB1 promoter to regulate the transcription of ITGB1.In addition,ITGB1 was knocked down in both SMMC-7721 and MHCC97H cells stably transduced with LV-C or LV-IER2,and these cells were subjected to cell spreading,Transwell migration and invasion to assess the requirement of ITGB 1 in IER2-promoted cell spreading,migration and invasion.(6)Western blot analyses were used to evaluate the effect of overexpression and knockdown of IER2 on the expression and activity of the ITGB 1-mediated downstream signaling molecules in SMMC-7721 and MHCC97H cells.Results:On the basis of SMMC-7721 and MHCC97H cell line,the the lentivirus-mediated IER2 stable or knockdown cells were established successfully.Through following assays,the effect of IER2 in migration,invasion and adhesion of hepatoma carcinoma cells was studyed initially.Results are:(1)Data from RT-qPCR and western blot analysis demonstrated that the HCC cell lines,HepG2,SMMC-7721,MHCC97H and HCCLM3 cells,four HCC cell lines with increasing spontaneous metastatic potential,were shown to express IER2 with relative low expression of IER2 in HepG2 and SMMC-7721 cells,which have low metastatic potential,and abundant expression of IER2 in MHCC97H and HCCLM3 cells,which have high metastatic potential.(2)Results from CCK-8 assays demonstrated that,compared with the corresponding empty vector-transduced cells and the nontransduced cells,knockdown and the ectopic expression of IER2 had no obvious effects on cell viability of SMMC-7721 and MHCC97H cells in vitro.(3)Transwell cell migration and invasion assays demonstrated that,compared with the corresponding empty vector-transduced cells and the nontransduced cells,both SMMC-7721 and MHCC97H overexpressing cells showed significant increase of the cell migratory and invasiveness capacity(p<0.05),whereas silencing of IER2 obviously attenuated the motility of SMMC-7721 and MHCC97H cells(p<0.05).(4)Results from HCC cell-ECM adhesion assay showed that,compared with those in the empty vector-transduced cells and the nontransduced cells,IER2 overexpression significantly promoted SMMC-7721 or MHCC97H cell adhesion onto the fibronectin,while IER2 knockdown decreased the cell adhesion onto the fibronectin(p<0.05).However,there was no significant alteration in the cell adhesion onto the collagen type I or Matrigel either in IER2 overexpressing cells or IER2 knockdown cells.(5)Cell spreading assay showed that,compared with those in the empty vector-transduced cells and the nontransduced cells,overexpression of IER2 significantly increased the ability of SMMC-7721 or MHCC97H cells to spread on the fibronectin coated surface(p<0.05),whereras IER2 depletion obviously decreased cell spreading on the fibronectin(p<0.05).(6)Data from RT-qPCR and western blot analysis demonstrated that IER2 overexpression or knockdown resulted in significant upregulation or downregulation of ITGB1,but not ITGA5 either in mRNA or protein levels in SMMC-7721 and MHCC97H cells.Meanwhile,Luciferase reporter gene assay showed that overexpression of IER2 markedly upregulated the luciferase activity of the ITGB1 promoter,while knockdown of IER2 significantly decreased the luciferase activity of the ITGB1 promoter in comparison with those in the nontransduced cells and empty vector-transduced cells.Furthermore,results from ChIP assay showed that IER2 may interact directly with ITGB1 promoter to regulate the transcription of ITGB1.In addition,our findings further demonstrated that ITGB1 knockdown in both SMMC-7721 and MHCC97H cells stably overexpressed IER2 or transduced with empty vectors significantly reduced cell spreading,migration and invasion.(7)Results from Western blot analysis showed that,compared with those in the empty vector-transduced cells and the nontransduced cells,overexpression of IER2 significantly upregulated pY397FAK,pY407FAK,pY576/Y577FAK,pY861 FAK,pY925FAK,pS910FAK,pY419Src and pY118paxillin(p<0.05),and decreased pY527Src,whereras knockdown of IER2 showed obvious decrease of pY527Src and increase of pY419Src.No significant differences of the expression of FAK,Src and paxillin were found either in the IER2 overexpressing cells or knockdown cells.Conclusion:(1)IER2 expression was correlated with the metastatic potential of HCC cell lines.(2)IER2 expression might promote the HCC cell migration,invasion,adhesion and spreading onto to fibronectin.(3)IER2 played a critical role in regulation of HCC cell-fibronectin adhesion,spreading,migration and invasion probably by the transcriptionally promoted ITGB1 and then activated ITGB1-FAK-Src-paxillin signal pathway.