The Expression Of PSMB4 In Hepatocellular Carcinoma And Its Effect On Proliferation And Apoptosis Of Hepatocellular Carcinoma Cell

Objective: This study was aimed to investigate the expression of the proteasome subunit beta type 4(PSMB4)in hepatocellular carcinoma(HCC),as well as the relationship between PSMB4 expression level and the clinicopathological parameters of HCC patients.In addition,we used specific short hairpin RNA(shRNA)to interfere with PSMB4 expression in HCC cells for further elucidating the effect of PSMB4 on the proliferation and apoptosis of HCC cells.Methods: We retrieved TCGA database to analyze the expression of PSMB4 in HCC primarily.Western blot was performed to detect the expression difference of PSMB4 in 8 HCC tissues and para-carcinoma tissues.Immunohistochemical staining was used to investigate the expression level of the PSMB4 in 105 HCC tissues and 25 normal liver tissues.Then the results of PSMB4 expression in HCC tissues were analyzed,as well as the relationship between its expression and clinicopathologic parameters,including gender,age,serum AFP level,cirrhosis,tumor number,HBV infection,maximum tumor diameter,tumor metastasis,microvascular invasion,tumor differentiation,TNM staging.The risk factors of survival of HCC patients were analyzed by univariate and multivariate analysis with Cox regression model.The relationship between PSMB4 expression level and survival of HCC patients was analyzed by TCGA database and Kaplan-Meier survival curve.Furthermore,the PSMB4 expression in normal liver cell line L02 and three HCC cell lines(HepG2,SMMC7721 and LM3)were tested.We detected the expression of PSMB4 in SMMC7721 cell lines after successfully transfecting with specific PSMB4 shRNA by western blot.And then the effects of proliferation and apoptosis of SMMC7721 cells in vitro after transfection of PSMB4 shRNA were further tested using MTT assay,plate cloning assay and flow cytometry.Results: It showed that the expression of PSMB4 mRNA was significantly higher in HCC tissues than in normal liver tissues on TCGA database(P<0.05).The western blot results revealed elevated PSMB4 expression in 7 tumor tissue samples compared with paired adjacent non-tumorous tissue samples(p<0.05),and there is no different expression in the other sample(P>0.05).In 105 cases of HCC,PSMB4 staining showed high expression in 54.3%(57/105)patients and low expression in 45.7%(48/105)patients,while most of normal liver tissues(68%)showed low or negative expression.Furthermore,immunohistochemical staining results showed that PSMB4 expression level in HCC tissues was markedly related to HBV infection,maximum tumor size,tumor differentiation,TNM staging.Univariate analysis showed that the survival in HCC patients was related to tumor size,metastasis,differentiation,TNM staging and PSMB4 expression level(P<0.05).Meanwhile,multivariate analysis showed that tumor metastasis and high PSMB4 expression were independent risk factors for HCC patients(P<0.05).TCGA database and Kaplan-Meier curve similarly revealed that patients with high PSMB4 expression had shorter survival time than those with low PSMB4 expression(P<0.05).On the other hand,the result of western blot showed that the expression of PSMB4 in HepG2 cells and SMMC7721 cells was significantly higher than that in normal liver L02 cells.In addition,the experimental group SMMC7721 cells that interfere with the expression of PSMB4 were successfully constructed and the interference efficiency was obvious(P<0.05).MTT assay,plate cloning assay and flow cytometry showed that the proliferation of SMMC7721 cells that interfered with PSMB4 expression was weakened and the apoptotic ability was enhanced(P<0.05).Conclusions: PSMB4 was markedly up-regulated in HCC tissues.The expression of PSMB4 was correlated with tumor size,histological grade and TNM staging.High expression of PSMB4 predicted an independent poor prognosis of HCC patients,suggesting that PSMB4 may serve as a potential prognosis marker or a treatment target.Depletion of PSMB4 inhibited HCC cell proliferation and promoted its apoptosis,implicating that PSMB4 may play a stimulative role in regulating the proliferation of HCC cells.