| Introduction:Hepatocellular carcinoma(HCC)is one of the most malignant cancer types worldwide,which causes over 700,000 deaths annually.Especially in east Asia,the incidence of HCC keeps high due to the severe infection of HBV or HCV,and our country takes over half of HCC patients of the whole world.Nowadays,there’re diverse diagnosis strategies towards HCC such as computed tomography(CT),magnetic resonance imaging(MRI)and ultrasonic contrast.Common therapeutic methods include hepatectomy,radiofrequency ablation,transcatheter arterial chemoembolization(TACE)and liver transplantation.Although significant improvements in diagnosis and treatment have been accomplished,the mortality rate for patients with HCC remains high.The rapid and unnoticeable development and metastases of HCC are crucial to its progression,resulting in unresectable vascular thrombi,intrahepatic satellite nodules or cancer recurrence.Hence,it is important to discover effective diagnostic biomarkers to identify HCC in the early stage to improve the prognosis of patients with HCC.Ubiquitination is a post-translational modification in which ubiquitin is enzymatically attached to the substrate protein,which then moves on to structured degradation;this process is collectively known as the ubiquitin-proteasome system(UPS).The UPS governs diverse cellular processes,including,but not limited to,cell cycle progression,cell death,and development.Abnormal ubiquitination affects cancer initiation and development.The UPS consists of three discrete enzymes: E1 ubiquitin-activating enzymes,E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases.The human genome encodes more than 600 E3 ligases,thereby providing substrate specificity in the UPS.E3 ligases are further categorized into three major groups: HECT,RING and PHD/U-box.The Skp1/Cul1/F-box-protein(SCF)E3ligase complex is the multi-subunit RING-type E3 ligase,which is the largest group of E3 ligases.To date,69 F-box proteins have been characterized in the human genome.Based on distinct domains,F-box proteins are categorized into FBXLs(leucine-rich repeats),FBXWs(WD40 motifs)and FBXOs(other domains),and thus,as recognition units,they are capable of processing specific substrates.The F-box protein β-Tr CP(also termed β-Tr CP1,F-box/WD repeat-containing protein 1A: FBXW1)regulates many cellular processes by targeting diverse substrates,and is reported to play oncogenic functions in cancers,yet the molecular mechanism of β-Tr CP within HCC remains to be elaborated.NF-κB inhibitor α(IκBα)is an isoform in the IκB family,members of which have multiple ankyrin repeat domains at the C-terminus.IκBα combines with the NF κB dimer to retain its existence in the cytoplasm and prevent its translocation into the nucleus.The IκB kinase(IKK)complex is induced by diverse extracellular signals,such as lipopolysaccharide,tumor necrosis factor(TNF)and growth factors,and the activated IKK complex phosphorylates IκBα,leading to degradation.While previous studies have reported its inhibition of NF-κB,the expression,function and relative molecular mechanism of IκBα in HCC are yet to be further elucidated.Containing 16 leucine-rich repeats and one PSD-95/Dlg/ZO-1(PDZ)domain,Erbin is a member of the leucine-rich repeat and PDZ domain family.Furthermore,Erbin is an adaptor of the Erb-b2 receptor tyrosine kinase 2(ERBB2)protein and,by binding with ERBB2,regulates its function and localization.Erbin has also been reported to be involved in several pathways.For instance,Erbin suppresses tumorigenesis via inhibiting Akt activity or Ras/Raf signaling.In addition,Erbin exerts an ontogenetic function by activating estrogen receptor(ER)α or inhibiting STAT3 signaling.Thus,there is conflicting data regarding the role of Erbin in cancer.A previous study showed that Erbin is upregulated following exposure of macrophages to muramyl dipeptide,TNF α and lipopolysaccharide.Although it has been observed that Erbin is upregulated in HCC tissues,the underlying mechanism via which Erbin becomes dysregulated remains unknown.According to our previous research mentioned above,in this study we intend to carry out assays,at both tissue and cell level,to explore the expression of β-Tr CP,IκBα and Erbin in HCC or para-cancerous tissues,the function they play in HCC cells,and the correlation or mechanisms of the interaction between the two proteins.Methods:1.Immunohistochemistry was employed to assess the expression of β-Tr CP,IκBα and Erbin in HCC samples acquired from 2011 to 2013 in tumor tissue bank of Hepatobiliary Surgery department of Southwest Hospital.The expression of proteins was analyzed with clinical characteristics of HCC patients to uncover the potential correlation.2.Huh7 and HCCLM3 cells were chosen as the representatives of HCC cells.Overexpression or knockdown of proteins were accomplished by transfection of plasmid or small interfering RNA.Western blot and real-time PCR were employed to detect the expression changes.3.Immunoprecipitation assay and in vivo ubiquitination assay were employed to detect the potential interactions among proteins.Cell counting kit(CCK)-8,colony formation assay,Transwell migration assay and relative Rescue experiments were used to evaluate the function of IκBα or Erbin in HCC cells.Results:1.Immunohistochemistry indicated that,compared with matched adjacent tumor tissues,the expression of β-Tr CP was increased in HCC tissues.Kaplan-Meier analysis suggested that patients with high expression survived for shorter time than those tested low or negative forβ-Tr CP(p=0.0253).CCK-8 assay and colony formation assay revealed that overexpression ofβ-Tr CP promoted proliferation of HCC cells,while down-regulation of β-Tr CP inhibited.Transwell migration,invasion and real-time PCR against markers of invasion demonstrated that overexpression of β-Tr CP enhanced metastasis of HCC cells,and down-regulation ofβ-Tr CP weakened.2.Western blot was used to detect the expression alteration of β-Tr CP substrates upon its regulation,which demonstrated that the expression of IκBα changed remarkably.Immunoprecipitation assay revealed that ectopically expressed β-Tr CP pulled down exogenous IκBα.In vivo ubiquitination assay illustrated that β-Tr CP promoted polyubiquitination of IκBα.Expression of IκBα,exposed of up-regulation of β-Tr CP,restored after treatment of MG132,an inhibitor of proteasome.The promoted proliferation and migration of HCC cells by overexpression of β-Tr CP were attenuated by up-regulation of IκBα or knock down of NF-κB p65.Immunohistochemistry revealed that compared with matched adjacent tumor tissues,the expression of IκBα was decreased in HCC tissues.Kaplan-Meier analysis suggested that patients with low expression survived for shorter time than those tested high for IκBα(p=0.0009).3.Western blot and real-time PCR collectively indicated that NF-κB promoted expression of Erbin at both transcription and translation process.CCK-8 and Transwell assays demonstrated that overexpression of Erbin significantly promoted proliferation and migration of HCC cells,whereas knockdown suppressed proliferation and migration.Reduction of Erbin expression attenuated the enhanced proliferation and migration abilities of HCC cells by NF-κB increasing.4.Compared with matched para-cancerous liver tissues,the expression level of Erbin was significantly increased in HCC tissues by immunohistochemistry.Kaplan-Meier analysis suggested that patients with high expression survived for shorter time than those tested low or negative for Erbin(p=0.0253).Erbin expression was associated with high TNM stage(p=0.039)and tumor recurrence(p=0.010)of HCC.The expression of β-Tr CP was negatively correlated with expression of IκBα(p<0.001)and positively with expression of Erbin(p<0.001).Conclusions:Expression of β-Tr CP increased in HCC tissues,and was negatively correlated with survival time of HCC patients.Up-regulation of β-Tr CP suppressed IκBα and activated NF-κB to induce overexpression of Erbin to promote proliferation and migration of HCC cells(β-Tr CP/IκBα/NF-κB/Erbin axis). |
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