Molecular Mechanism And Function Of MT1E Gene In Hepatocellular Carcinoma

Background and objectives:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide.Despite the awareness-raising,early detection,and effective preventive measures in various countries in recent years,the incidence rate and mortality rate of HCC are still rising.At present,surgery is the main treatment for HCC,combined with radiotherapy and chemotherapy,targeted therapy,and other comprehensive treatment methods.However,most patients will eventually develop into metastatic diseases and die of this cancer.Therefore,new methods for early diagnosis,treatment,inhibition of tumor progression,and effective control of this disease still need to be further explored.It may be one of the breakthrough points of molecular targeted therapy for liver cancer in the future.Recent studies have reported that metallothioneins(MTs)are involved in the occurrence and prognosis of a variety of tumors,including HCC.As a member of the MTs family,the biological function and methylation status of MT1E in HCC remains to be elucidated.The purpose of this study is to explore the expression and function of MT1E in HCC and its epigenetic regulation mechanism,which provide new approach for clinical diagnosis,treatment and prognosis of HCC.Methods:1.We analyzed the differentially expressed genes(DEGs)between tumor and normal tissues in the tumor Genome Atlas(TCGA)and genotype tissue expression(GTEx)database,and determined the expression level of MT1E in HCC,2.qPCR and Western blot analyses were applied to determine the expression level of MT1E in HCC and adjacent normal tissues,and then the relationship between MT1E and clinical parameters of HCC was further analyzed.3.We applied the MethPrimer to predict the CpG islands of MT1E promoter region,and examined the expression and methylation status of MT1E in HCC tissues and cells by qPCR,MSP,and BSP.4.The pEGFP-N1-MT1E and pEGFP-N1 empty vectors were transfected into Hep3B and SMMC7721 cells.si-MT1E and non-specific siRNA double strand were transfected into HCCLM3 cells.MTT and colony formation assays were applied to evaluate the cell viability and proliferation of HCC.Besides,transwell assay was employed to determine the abilities of cell migration and invasion of HCC.The effectd of MT1E on the apoptosis rate and cell cycle distribution of HCC cells were examined by flow cytometry.5.Western blot was used to evaluate the expression levels of some related markers of EMT in HCC cell lines before and after transfection.Results:1.MT1E gene was screened out by bioinformatics analysis in TCGA and GTEx database.2.We determined that MT1E expression was down-regulated in HCC tissues by western blot and qPCR,which was associated with TNM stage and differentiation of HCC,but not to gender,age,tumor size,HBV infection and liver cirrhosis.The expression of MT1E was significantly correlated with the abnormal methylation level of gene promoter.3.The expression levels of MT1E in Hep3B,SMMC7721,HCCLM3,and HepG2 cells and L02 cells were detected by semi-quantitative RT-PCR,exhibiting that MT1E expression was lost in Hep3B,SMMC7721,and HepG2 cells,but decreased in HCCLM3 cells.The expression of MT1E was restored in Hep3B,SMMC7721,and HepG2 cells treated with 5-Aza-dC,but increased in HCCLM3 cells.4.Methprimer software predicted that there were obvious CpG islands in the MT1E promoter region;MSP analysis detected the methylation level of MT1E in its promoter region.Our results demonstrated that partial methylation was observed in Hep3B,SMMC7721,HCCLM3,and HepG2 cells,but no methylation was found in LO2 cells.5.The methylation status of MT1E promoter in Hep3B,SMMC7721,and HCCLM3 cells were detected by BSP,and the methylation level of MT1E in HCC and matched normal tissues were examined by MSP.Our results demonstrated that the methylation levels of MT1E in HCC tissues were observably higher than that in matched normal tissues.6.MTT assay showed that the viability of Hep3B and SMMC7721 cells reduced markedly after MT1E overexpression.In HCCLM3 cells,MT1E knockout increased cell viability.Colony formation results assay indicated that the number of clones was observably decreased after MT1E overexpression in Hep3B and SMMC7721 cells.Knockdown of MT1E in HCCLM3 cells notably elevated the number of clones.7.The effects of MT1E on cell apoptosis and cell cycle distribution of HCC cells were detected by flow cytometry.Our data displayed that MT1E overexpression significantly increased the apoptosis rate of Hep3B and SMMC7721 cells and induced the arrest of Hep3B and SMMC7721 cells in G0-G1 phase,while MT1E knockdown markedly decreased the apoptosis rate of HCCLM3 cells and induced the arrest of HCCLM3 cells in S phase.8.The effects of MT1E on the tumorigenicity of HCC cells were detected in nude mice.The results showed that overexpression of MT1E in Hep3B cells significantly reduced tumor weight and volume.9.The effects of MT1E on HCC cell migration and invasion were measured by transwell assay.Our data exhibited that in Hep3B and SMMC7721 cells,MT1E overexpression markedly decreased the number of migration and invasive cells.However,in HCCLM3 cells,MT1E knockdown dramatically enhanced the number of migration and invasive cells.The above results suggested that MT1E could dramatically suppress the migration and invasion of HCC cells.10.Western bolt demonstrated that after MT1E overexpression,the expression of E-cadherin enhanced,while the expression of N-cadherin,vimentin and snail reduced in Hep3B and SMMC7721 cells.In HCCLM3 cells,after MT1E knockout,the expression of E-cadherin decreased,while the expression of N-cadherin,vimentin and snail increased.The above results showed that MT1E inhibited the occurrence of EMT in HCC cells,which resulted in decreased migration and invasion.Conclusions:The down-regulation of MT1E expression in HCC is due to the epigenetic silencing of MT1E caused by promoter methylation.In HCC cells,MT1E can markedly suppress the proliferation,migration,invasion,cell cycle progression,and tumorigenicity of HCC cells in vivo,and induce apoptosis of HCC cells and inhibit the occurrence of EMT transformation.Therefore,MT1E can be used as a new molecular marker of HCC and a potential therapeutic target.