| Background and Object:Hepatocellular carcinoma(HCC)is one of the most prevalent malignancies and accounts for the second leading cause of cancer-related death.One of the greatest problems preventing potential curative treatment for HCC,including ablation,transplantation and resection,is the high recurrence rate.Furthermore,surgical intervention is not feasible for the majority of HCC patients with intermediate-or advanced-stage disease,while chemotherapy and radiotherapy have exhibited only limited efficacy.Therefore,a better understanding of the molecular mechanisms underlying HCC development may reveal possible pathogenic and therapeutic implications and help improve the overall survival of HCC patients.[Su(var)3-9,Enhancer-of-zeste and Trithorax] domain-containing protein 7(SET7/9),coding gene located in human chromosome 4q28,is a member of the protein lysine methyltransferase(PLMT)family that methylates both histone H3K4 and some other nonhistone proteins including transcription factors,tumor suppressors and membrane-associated receptors.The full-length mRNA of transcription is 7374 nt,the length of the CDS coding region is 1098 nt,and the encoded protein is composed of 366 amino acids.In recent years,several studies have found that protein lysine methylation is a pivotal method of posttranslational modification,which can affect cell growth,proliferation,and apoptosis.Changes in the expression level of SET7/9 are frequently detected in various types of human cancer.SET7/9 can regulate the activities of its protein targets both positively or negatively,thus leading to a serise of cellular effects related with cancer initiation and development.However,so far the function of SET7/9 in HCC has remained unknown.This study aims to investigate the role of SET7/9 in HCC,evalute the effects of SET7/9-mediated methylation on HCC initiation and development,and unravel the underlying molecular mechanisms involving the function of SET7/9.The transcription factor E2F1 is a member of the E2F(E2 promoter-binding factor)family,and its coding gene is located on human chromosome 20q11.The mRNA produced by transcription is 2486 bp in length and the encoded protein is composed of 437 amino acids.E2F1 regulates multiple gene expression and affects DNA synthesis,cell cycle,and apoptosis.When promoting apoptosis,the p53-dependent and p53-independent cellular signaling pathways activate downstream pro-apoptotic proteins such as Caspase-3 and Caspase-9.Lysine methylation at position 185 of the E2F1 peptide chain inhibits acetylation and phosphorylation of the protein,promotes its ubiquitination and inhibits apoptosis.Overexpression of E2F1 can both drive cells into S phase and induce apoptosis,and its regulation of cell cycle and apoptosis depends on its modification type and stability.Therefore,E2F1,as an important transcriptional activator,can promote cell cycle progression and cell proliferation,and inhibition of its activity is expected to be an effective means to control tumor pathogenesis.Therefore,to clear that the effect and mechanism of SET7/9regulation of E2F1 methylation modification in hepatocellular carcinoma cells is the focus of this paper.Methods:This study was carried out in three main aspects: clinical pathological analysis,cell functional experiments,and identification of the underlying molecular mechanisms.1.Clinical pathological analysis of differential SET7/9 expression in hepatocelluar carcinoma tissue and para-carcinoma normal tissueDifferential expression of SET7/9 proteins in tumor tissues and para-carcinoma normal liver tissues were examined with immunohistochemical assay.Statistical analyses were performed to detect the significance of differences between SET7/9 expression and various pathological parameters including age,gender,TNM stage,and pathological stage of 68 HCC patients.Using western blot analyses,the expression levels of SET7/9 in HCC tumor tissues and the paired normal tissues were examined and the expression differences were compared statistically.2.Clinical pathological analysis of differential E2F1 expression in hepatocelluar carcinoma tissue and para-carcinoma normal tissueDifferential expression of E2F1 proteins in tumor tissues and para-carcinoma normal liver tissues were examined with immunohistochemical assay.Statistical analyses were performed to detect the significance of differences between E2F1 expression and various pathological parameters including age,gender,TNM stage,and pathological stage of 68 HCC patients.Using western blot analyses,the expression levels of E2F1 in HCC tumor tissues and the paired normal tissues were examined and the expression differences were compared statistically.3.Cell functional assay of HCC cells with aberrant SET7/9 or E2F1 expression and its relationship with methylationSET7/9 expression in four HCC cell lines SMMC-7721,Bel-7404,HepG2,and Huh7 and one human normal liver cell line LO2 were examined using western blot analyses and two HCC cell lines were selected for further experiment.SET7/9-overexpressed and-silenced vectors were constructed and transfected into the corresponding HCC cell lines.The overexpression and silencing efficiencies were examined using western blot analyses.The cell proliferation ability,plate colony formation ability,invasive ability,and migratory ability of HCC cells with overexpressed-or silenced-SET7/9 expression as well as the control HCC cells were tested using the CCK8 assay,transwell migration assay,and wound healing assay,respectively.E2F1-overexpressed and-silenced vectors were constructed and transfected into the corresponding HCC cell lines.The overexpression and silencing efficiencies were examined using western blot analysis.The cell proliferation ability,plate colony formation ability,invasive ability,and migratory ability of HCC cells with overexpressed-or silenced-E2F1 expression as well as the control HCC cells were tested using the CCK8 assay,transwell migration assay,and wound healing assay,respectively.The experiment results of SET7/9-and E2F1-silenced cells were compared with the results of cells treated with 5'-deoxy-5'-methylthioadenosine(MTA)to examine the effects of expression change in SET7/9 or E2F1 as well as MTA treatment on cell function.4.Investigation of the molecular mechsniams underlying SET7/9-mediated E2F1 methylation in the initiation and development of HCCQuantitative real-time PCR(qRT-PCR)and western blot analyses were used to examine changes of E2F1 expression at the mRNA and protein levels before and after SET7/9 over expression or silencing,as well as changes of SET7/9 expression at the mRNA and protein levels before and after E2F1 expression or silencing.Subcellular fractionation and coimmunoprecipitation analyses were performed to investigate the interaction between SET7/9and E2F1 proteins.Potential down-stream targets along the SET7/9-E2F1 axis related to SET7/9 function were searched from literature and public database.Expression differences of these key downstream protein factors in SET7/9-overexpressed or-silenced cells and the control cells were examined using western blot analyses in order to validate the effect of SET7/9-mediated E2F1 methylation on downstream signaling pathways.5.Statistical analysisAll statistical computations were performed using SPSS software version 20.Correlations between SET7/9 and E2F1 expression and various clinicopathological parameters were evaluated with the ?2 test.The significance of difference in the expression levels of SET7/9 and E2F1 between HCC tumor tissues and the paired normal tissues was evaluated with paired t-test.The cell proliferative ability,cell migratory rates,cell invasive ability,and the expression levels of SET7/9 and E2F1 were compared using one-way analysis of variance(ANOVA)followed by Tukey's post-hoc test among the SET7/9-or E2F1-overexpressed group,SET7/9-or E2F1-silenced group,the MTA-treated group,and the corresponding control group.P-value <0.05 was considered to indicate statistically significant differences.Results:1.Expression of SET7/9 in clinical HCC tissuesAmong the 68 clinical samples of HCC tunor tissue,SET7/9 exhibited high expression in 53 cases(77.94%).The expression level of SET7/9 in clinical HCC samples was significantly correlated with tumor dimension(P<0.05)and pathological stage(P<0.05).However,no significant correlation was detected between the expression level of SET7/9 and age,gender,lymph node metastasis,and distant metastasis.2.Expression of E2F1 in clinical HCC tissuesE2F1 exhibited high expression in 44 cases(64.71%)of all the 68 clinical HCC tissues.The expression level of E2F1 in clinical HCC samples was significantly associated with tumor dimension(P<0.05),lymph node metastasis(P<0.05)and pathological stage(P<0.05).However,no significant correlation was detected between the expression level of E2F1 and age,gender,and distant metastasis.3.Effects of aberreant SET7/9 and E2F1 expression on cellular behavior of HCC cells and the relationship with methylationWestern blot analysis revealed varied expression levels of SET7/9 protein in four HCC cell lines(SMMC-7721,Bel-7404,HepG2,and Huh7)and one normal liver cell line(LO2).Among the four HCC cell lines,SET7/9 showed the lowest expression in Bel-7404 and HepG2 and the highest expression in Huh7 and SMMC-7721.However,all the four human HCC cell lines exhibited higher SET7/9 expression as compared with the normal liver cell line LO2.Considering the comprehensive conditions during cell culture and the expression levels of SET7/9 in different HCC cell lines,HCC cell line Huh7 was used for constructing shRNA-mediated SET7/9-silenced cell line,while cell line Bel-7404 was used forconstructing SET7/9-overexpressed cell line.SET7/9-overexpressed and-silenced HCC cell lines Bel-7404-SET7/9 and Huh7-shRNA-SET7/9 were constructed using vector transfection.Western blot and qRT-PCR analyses confirmed a significant increase of SET7/9 expression in the Bel-7404-SET7/9 cells and a decrease of SET7/9 expression in the Huh7-shRNA-SET7/9 cells.Bel-7404-SET7/9cells showed significantly enhanced proliferation,invasion,and migration compared with the control cells as determined by the CCK8 assay,wound healing assay,and transwell migration assay(p < 0.05).On the contrary,Huh7-shRNA-SET7/9 cells showed significantly reduced proliferation,invasion,and migration compared with the control cells as determined by the CCK8 assay,wound healing assay,and transwell migration assay(P<0.05).E2F1-overexpressed and-silenced HCC cell lines Bel-7404-E2F1 and Huh7-shRNAE2F1 were constructed using vector transfection.Western blot and qRT-PCR analyses confirmed a significant increase of E2F1 expression in the Bel-7404-E2F1 cells and a decrease of E2F1 expression in the Huh7-shRNA-E2F1 cells.Bel-7404-E2F1 cells showed significantly enhanced proliferation,invasion,and migration compared with the control cells as determined by the CCK8 assay,wound healing assay,and transwell migration assay(P<0.05).On the contrary,Huh7-shRNA-E2F1 cells showed significantly reduced proliferation,invasion,and migration compared with the control cells as determined by the CCK8 assay,wound healing assay,and transwell migration assay(P<0.05).The effects of SET7/9-or E2F1-silencing on HCC cells were similar to the effects of MTA-treatment,reflecting a potential realtionship between changes in SET7/9 or E2F1 expression and protein methylation.4.The regulatory relationship between SET7/9 and E2F1 and the molecular mechanisms underlying SET7/9-mediated E2F1 methylation in the initiation and development of HCCWestern blot and qRT-PCR analyses showed that expression or silencing of SET7/9 in Huh7 cells did not affect E2F1 expression at the mRNA level,but decreased E2F1 expression at the protein level.Treatment of Huh7 cells with MTA(5'-deoxy-5'-methylthioadenosine),an inhibitor of protein methylation,also led to the similar changes in E2F1 protein expression as compared with the SET7/9-silenced group.However,no evident change in SET7/9 expression at either the mRNA or protein level was observed after E2F1 expression or silencing.Coimmunoprecipitation analysis revealed protein-protein interaction between SET7/9 and E2F1 in HCC cells.Subcellular fractions derived from Huh7 HCC cells were further analyzed for the location of interaction between SET7/9 and E2F1.Direct interaction of these two proteinswas detected in the cytoplasm,while no interaction was detected in the nucleus.Using western blot analyses,we also investigated the downstream activation of cyclin A2,cyclin E1,and CDK2 in SET7/9-overexpressed and-silenced HCC cells.Increased cyclin A2,cyclin E1,CDK2,and E2F1 abundance was observed in SET7/9-overexpressed cells,while the opposite effects were observed in SET7/9-silenced cells.Conclusion:1.SET7/9 was up regulated in HCC and the expression level of SET7/9 was significantly associated with tumor dimension and pathological stage.2.E2F1 was up regulated in HCC and the expression level of E2F1 was significantly associated with tumor dimension,lymph node metastastsis,and pathological stage.3.SET7/9 promotes HCC cell proliferation,invasion,and migration through methylation of E2F1 at the translational level.4.SET7/9-mediated methylation of E2F1 affects the expression levels of E2F1 downstream targets cyclin A2,cyclin E1,and CDK2,thus regulating the initiation and progression of HCC.Innovation:1.This study reveals the expression levels of SET7/9 and E2F1 in clinical HCC tissues and the clinical significance.2.This study reveals the roles of SET7/9 and E2F1 in regulating HCC cell proliferation,invasion,and migration.3.This study identifies E2F1 as an important methylation target of SET7/9 and unravels the underlying molecular mechanisms of SET7/9 in promoting HCC development and metastasis through methylation of E2F1. |
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