The Role Of Coatomer Protein Complex Subunit β’(COPB2) And Its Potential Mechanism Underlying Cell Proliferation And Apoptosis In Cholangiocarcinoma

Cholangiocarcinoma is the second most common primary malignant tumor of the liver and gallbladder system,derived from bile duct epithelial cells,characterized with the onset of concealment,easy to early infiltration and distal metastasis,poor prognosis and so on.According to the anatomy of cholangiocarcinoma,it can be divided into intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma.Radical resection is the only effective way to treat cholangiocarcinoma.But because of its onset concealment,rapid progress,nonsensitive to radiotherapy and chemotherapy and other characteristics,clinical efficacy treatment of cholangiocarcinoma is poor,and the five-year survival rate is no more than 10%.The primary cause is that the molecular mechanism of the development of cholangiocarcinoma is not entirely clear.Therefore,it is pivotal to screen the new core molecules in the process of the development of cholangiocarcinoma,and to clear the molecular mechanism of cholangiocarcinoma.This will be helpful for the target therapy to improve the survival rate of patients with cholangiocarcinoma.COPB2 gene,the coatmoter protein complex subunitβ’,is located at 3q2.3,and encodes theβ’-COP subunit and is one of the subunits of the coatmoter protein complex,which involves in the retrograde traffic of the vesicles between the endoplasmic reticulum and the Golgi apparatus.Studies have shown that COPB2interacts with ADP-ribosylation factor（ARF）GTPase-activating protein 2（GAP2）and KKXX-containing molecules to complete its normal physiological function.In addition,COPB2 also activates cell growth signaling and induces cell mitosis via interacting with beta-receptors of platelet-derived growth factor（PDGF）,.COPB2 expression is also associated with tumor.The study found that COPB2was involved in cell growth and cell apoptosis in mesothelioma cells.In addition,COPB2 was also abnormally highly expressed in the tumor tissue of the patients with prostate cancer and prostate cancer cell lines LnCaP,DU-145,PC-3 and CWR22RV1.COPB2 silencing not only inhibited PC-3 cell proliferation and clonal formation,but also promoted apoptosis,and cell cycle arrest in S phase.However,COPB2 function in cholangiocarcinoma is not clear.In view of this,this present study will explore the role of COPB2 in cholangiocarcinoma cell proliferation,apoptosis and the possible molecular mechanism.【Objective】1.To detect the expression of COPB2 and to analyse its clinical significance in cholangiocarcinoma.2.To determine the possible effect of COPB2 on cellular proliferation and apoptosis in cholangiocarcinoma.3.To investigate the possible molecular mechanism of COPB2 in promoting the proliferation of cholangiocarcinoma cells and to identify potential key molecules.【Methods】1.Clinical samples of 27 patients with cholangiocarcinoma were collected including 9 case with cancer-adjacent normal tissues,together with corresponding clinical data including sex,age,lymph node metastasis and TNM stage,histopathological grade.Immunohistochemistry was used to explore the expression of COPB2 in cholangiocarcinoma and its cancer-adjacent normal tissues.The positive rate and staining intensity of stained cells were scored.The result of multiplification with"the score of positive rate of stained cells"and"the score of staining intensity"was assessed as the total score:（1）0-1 as low expression;（2）≥2 as high expression.Fisher’s exact test and McNemar test were used to analyze the correlation between the clinical significance and expressions of COPB2.2.Firstly,qPCR was used to explore the expression of COPB2 gene in two cholangiocarcinoma cell lines,including RBE and QBC939.Secondly,the fragment for COPB2-specific interference was confirmed on the basis of the COPB2 nucleotide sequence from the public NCBI database.The corresponding control interference（shCtrl）and the COPB2-specific interferred lentivirus（shCOPB2）vectors were constructed by GV115 lentivirus.shCtrl or shCOPB2 lentivirus was transfected into293T cells respectively.The efficiency of COPB2 knockdown was tested by using Western blot and real-time quantitative PCR.MTT assay and Celigo method were applied to observe the growth viability of RBE and QBC939 cells continously for 5 days.RBE and QBC939 cells transfected with shCOPB2 or shCtrl vector were stained with propidium iodide or Annexin V-APC staining kit.The number of apoptotic cells at early stage was checked by flow cytometry to analyze the effect of COPB2 knockdown on apoptosis of RBE and QBC939 cells.The proportion of cells at G₂/M,S and G₀/G₁ phages was determined by flow cytometry for analyzing the effect of COPB2 knockdown on cell cycle.3.Total protein was extracted from RBE and QBC939 cells transfected with shCtrl or shCOPB2.PathScan?RTK Signaling Antibody Array Kit was used to incubate with COPB2 primary antibody.Data from shCtrl group and shCOPB2 group were extracted to analyze differentially expressed molecules.【Results】1.Immunohistochemistry revealed that COPB2 was highly expressed in the cells in cancer than that in adjacent normal tissues.Statistical results showed that the expression of COPB2 was connected with pathological,TNM stage,grade tumor size,and lymph node metastasis（P<0.05,respectively）,but was not related to age,gender（P>0.05,respectively）.Compare to normal bile duct tissues,the expression of COPB2 in cholangiocarcinoma tissues was significantly higher by Fisher’s Exact test（P=0.008）.2.The results of qPCR revealed that COPB2 was highly expressed in cholangiocarcinoma cell lines RBE and QBC939.PCR and sequencing revealed that the target sequence of COPB2 was successfully transfected into the vector GV115.The normal growth of 293T cells transfected with shCtrl or shCOPB2 lentivirus was not impacted.Compare to shCtrl group,qPCR showed that the expression of COPB2mRNA was significantly lower in shCOPB2 group（P<0.01）.Western blot confirmed that COPB2 protein level in shCOPB2 group was significantly lower than that in shCtrl group.Compared with the shCtrl group,MTT assay and Celigo counting certified that the cell proliferation was significantly inhibited in the shCOPB2 group（P<0.01）,especially on the 4th day and the 5th day.The results above shows that knockdown of COPB2 affects the proliferation of RBE and QBC939 cells.Compared to shCtrl group,the percentage of cells in G₀/G₁ phase of shCOPB2 group was significantly increased（P<0.01）,while the percentage of cells in S phase was significantly decreased（P<0.01）.However,the percentage of cells in G₂/M phase were not obviousely different in RBE and QBC939 cells.This suggests that COPB2knockdown in RBE and QBC939 cells led to cell cycle arresting at G₀/G₁ phase.Apoptosis assay indicated that the percentage of apoptotic cells in shCOPB2 group was significantly increased than that in shCtrl group,（P<0.01）.3.Analysis of antibody array showed that compared to shCtrl group,13molecules in shCOPB2 group were expressed differentially,including 6down-regulated genes and 7 up-regulated genes,which the most significant up-regulated molecule is ERK1/2（up to about 39.4%）and the most significant down-regulated molecule is Src（down about 25.5%）.The result indicated that shCOPB2 inhibited the proliferation of cholangiocarcinoma cells mainly through activiting ERK1/2 and inhibiting Src.【Conclusion】1.Compare with cancer-adjacent normal tissues,the expression of COPB2 in cholangiocarcinoma tissues was significantly higher.The correlation analysis revealed that COPB2 was statistically related to TNM stage,tumor size,lymph node metastasis and pathological grade,but not related to sex,age.2.COPB2 knockdown significantly inhibits the proliferation of cholangiocarcinoma cells,promotes their apoptosis,resultes in cell cycle arrest in G₀/G₁ phase.3.Inhibition of cholangiocarcinoma cells induced by COPB2 knockdown may due to activate ERK1/2 and supress Src.