| Purpose:This study aims to investigate the expression,underlying biological function and clinical significance of coatomer protein complex subunit beta 2(COPB2)in hepatocellular carcinoma(HCC).Methods:Hepatocellular carcinoma(HCC)mRNA expression data from the Tumor Genome Atlas(TCGA)database,International Cancer Genome Consortium(ICGC)database and Gene Expression Omnibus(GEO)database was used to analyze the differences of COPB2 expression in HCC tissues and corresponding non-tumor tissues.The cancer resection specimens and corresponding paracancerous specimens of 20 patients with hepatocellular carcinoma who were admitted to the Provincial Hospital of Shandong University from June 2019 to August 2020 were used to verify the difference of COPB2 expression at the protein level with immunohistochemical staining.After combining the clinical data and mRNA expression data of patients with hepatocellular carcinoma from the TCGA database and ICGC database,a logistic regression model was used to analyze the differences expression of COPB2 distribution in various clinicopathological characteristics;Univariate and multivariate Cox risk proportional regression model and Kaplan-Meier method were used to analyze the relationship between COPB2 expression and patient prognosis;Gene set enrichment analysis(GSEA)was used to explore the underlying pathways that might related to the high expression of COPB2 in HCC.Two hepatocellular carcinoma cell lines,BEL7402 and SMMC7721,were selected,and COPB2 were down-regulated in these two cell lines with siRNA.Scratch wound healing assays,transwell assays,CCK-8 assays,and cell cycle distribution measurement were performed to detect the effects of proliferation,migration and invasion;western blotting assays was used to detect the changes in the expression of epithelial-mesenchymal transition(EMT)related proteins,E-cadherin,N-cadherin,Vimentin and Snail,as well as mTOR pathway proteins,mTOR,p70 S6K,p-mTOR,p-p70 S6K and Cyclin D1 to explore the potential mechanism.Results:1.COPB2 expression increased in HCC:COPB2 mRNA expression in HCC tissues from TGCA cohort,ICGC cohort,GES76427 data set and GSE14520 data set were all significantly higher than the corresponding adjacent non-tumor tissues(all p<0.0001);immunohistochemical results showed that the COPB2 protein expression in HCC tissues was significantly higher than that in adjacent non-tumor tissues(p<0.0001).2.High expression of COPB2 was positively associated with poor prognosis in patients with HCC:COPB2 mRNA expression was significantly associated with higher AFP(TCGA:>20 vs.≤20,OR=1.616,95%CI:1.006-2.60,p<0.05)and higher T stage(TCGA:T3+T2 vs.T1,OR=1.539,95%CI:1.013-2.345,p<0.05),worse pathological stage(TCGA:Ⅲ vs.Ⅰ,OR=1.744,95%CI:1.033-2.969,p<0.05;ICGC:Ⅳ vs.Ⅰ,OR=3.833,95%CI:1.216-13.272,p<0.05,Ⅲ+Ⅳ vs.Ⅱ+Ⅰ,OR=1.796,95%CI:1.056-3.081,p<0.05)and poorer histological grade(TCGA:G4+G3 vs.G2+G1,OR=1.746,95%CI:1.136-2.695,p<0.05;ICGC:G2 vs.G1,OR=2.685,95%CI:1.182-6.566,p<0.05,G3 vs.G1,OR=3.889,95%CI:1.570-10.291,p<0.01);Kaplan-Meier survival analysis showed that the prognosis of COPB2 high expression group was worse(TCGA:HR=2.478,p<0.0001;ICGC:HR=1.848,p<0.05);univariate COX(HR=1.068,p<0.0001)and multivariate COX(HR=2.011,p<0.05)regression analysis suggests that COPB2 is an independent risk factors for the prognosis of HCC.3.Gene enrichment analysis(GSEA):cell cycle(NES:2.114,FDR:0.001,p<0.001),ERBB(NES:2.065,FDR:0.002,p<0.001),VEGF(NES:2.011,FDR:0.002,p<0.001),WNT(NES:2.008,FDR:0.001,p<0.001),mTOR(NES:1.997,FDR:0.002,p<0.001),NOTCH(NES:1.953,FDR:0.002,p<0.001),MAPK(NES:1.941,FDR:0.002,p<0.001),P53(NES:1.869,FDR:0.005,p<0.01)and TGF-β(-NES:1.784,FDR:0.012,p<0.01)signal pathway enrichment in the COPB2 high expression group.4.Downregulation of COPB2 inhibit the migration(BEL7402,wound closure tate,siNC:siCOPB2=[36.16±5.79%]:[18.99±2.21%],p<0.001,transwell migration,siNC:siCOPB2=[270.40±20.27]:[117.7±12.93],p<0.001;SMMC7721,wound closure tate,siNC:siCOPB2=[52.26±4.61%]:[24.38±5.96%],p<0.01;Transwell migration,siNC:siCOPB2=[301.67±35.60]:[117.89±18.77],p<0.0011)and invasion(BEL7402,Transwell invasion,siNC:siCOPB2=[206.60±14.02]:[72.89±13.14],p<0.00];SMMC7721,Transwell invasion,siNC:siCOPB2=[226.00±21.26]:[56.00±17.97],p<0.001)ability of HCC cell lines and make the epithelial-mesenchymal transition related protein N-cadherin(BEL7402,p<0.001;SMMC7721,p<0.001),Vimentin(BEL7402,p<0.001;SMMC7721,p<0.001)and Snail(BEL7402,p<0.001;SMMC7721,p<0.001)expression decreased,E-cadherin(BEL7402,p<0.01;SMMC7721,p<0.01)increased expression.5.Downregulation of COPB2 inhibit the proliferation rate(BEL7402,24h,siNC:siCOPB2=[1.74±0.13]:[1.28±0.19],p<0.01,48h,siNC:siCOPB2=[3.18±0.16]:[1.89±0.25],p<0.0001,72h,siNC:siCOPB2=[5.30±0.22]:[3.21±0.21],p<0.0001;SMMC7721,24h,siNC:siCOPB2=[1.27±0.18]:[0.92±0.15],p<0.01,48h,siNC:siCOPB2=[2.19±0.21]:[1.45±0.18],p<0.0001,72h,siNC:siCOPB2=[4.18±0.27]:[2.43±0.31],p<0.0001)and cause the G0/G1 phase arrest of cell cycle(BEL7402,siNC:siCOPB2=[42.87±2.37%]:[64.97±2.44%],p<0.001;SMMC7721,siNC:siCOPB2=[58.97±1.76%]:[72.70±2.21%],p<0.001)in HCC cell lines.The expression of the downstream proteins of mTOR pathway,mTOR(BEL7402,p<0.01;SMMC7721,p<0.01),p70 S6K(BEL7402,p<0.001;SMMC7721,p<0.001),phospho-mTOR(BEL7402,p<0.01;SMMC7721,p<0.01),phospho-p70 S6K(BEL7402,p<0.001;SMMC7721,p<0.001)and cyclin D1(BEL7402,p<0.001;SMMC7721,p<0.001),were also decreased with the downregulation of COPB2.Conclusion:COPB2 is a novel prognostic biomarker and a promising therapeutic target for HCC. |
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