Analysis Of The Expression Of ANXA5 In Intrahepatic Cholangiocarcinoma Tissure And Study Of Biological Effects Of ANXA5-shRNA On QBC939 Cells

Part 1 Analysis of the expression of ANXA5 in human intrahepatic cholangio-carcinoma tissureObjective To study the expression of Annexin A5(ANXA5)in intrahepatic cholangiocarcinoma(ICC)tissure and its relationship with clinicopathological parameters,and the effect of high expression of ANXA5 on survival rate.Methods According to the inclusion criteria,86 patients with ICC from June 2006 to June 2014 were enrolled from Jiangsu Province Hospital and Northern Jiangsu People’s Hospital.The expression of ANXA5 in ICC tissues and correspondingadjacent tissues was studyed by Western Blotting and immunohistochemistry.The relationship between the expression of ANXA5 and the Clinicopathological features of ICC was studied by using statistical methods.Survival rate between ANXA5-positive and-negative patients was compared based on follow-up results.Results The expression of ANXA5 protein in ICC was significantly higher than that in the corresponding adjacent non-cancerous tissue(P<0.05).The expression of ANXA5 protein was correlated with tumor differentiation,lymph node metastasis and portal vein tumor thrombus in ICC(P<0.05),but not with the size of the tumor(P>0.05).Patients with high expression of ANXA5 had a shorter survival than those with low expressionConclusion ANXA5 may play an important role in the carcinogenesis and progression of ICC,which may provide a new way for gene therapy of ICC.Patients with high expression of ANXA5 had a shorter survival than those with low expressionPart 2 Construction and packaging of ANXA5-shRNA lentiviral vectorObjective The expression of ANXA5 in two human cholangiocarcinoma cell lines QBC939 and RBE was compared.QBC939 cells with high expression of ANXA5 were selected to enter RNAi experiments.ANXA5-shRNA lentiviral vector was constructed and transfected into 293T cells to produce LV-ANXA5-RNAi lentivirus.The QBC939 cells transfected by the lentivirus were used to knock down the expression of ANXA5.Methods Three types of ANXA5-shRNA oligodeoxynucleotides were constructed,and the three plasmids psc16759-1,psc16760-1 and psc16761-1 were constructed by lentiviral vector of GV248.Then 293T cells(lentivirus packaging cells)were infected and the three viruses LVpsc16759-1,LVpsc16760-1,LVpsc16761-1 were harvested.QBC939 cells were infected with the three viruses,and high titer virus was screened by qPCR.Results Three ANXA5-shRNA viruses,LVpscl6759-1,LVpsc16760-1 and LVpsc16761-1,were obtained.The knockdown rates of ANXA5 were 89.2%,77.1%and 32.9%respectively.The knockdown efficiency of the ANXA5 gene of the LVpsc16759-1 virus reached 89.2%,and the virus could be used for subsequent cell functional studies.Conclusion The lentiviral vector of ANXA5-shRNA was successfully constructed.After LVpsc16759-1 lentivirus transfecting into QBC939 cells,the knockdown efficiency of the ANXA5 gene was high.And the virus could be used in the next experiment.Part 3 Inhibitory effect of ANXA5-shRNA on QBC939 cells in vitroObjective The ANXA5 protein in QBC939 cells was down-regulated by ANXA5-shRNA interference,and the biological effects of QBC939 cells were investigated.Methods In the experiment,three groups of cells were set up:blank control group(BC group,QBC939),negative control group(NC group,QBC939-con077)and knockdown group(KD group,QBC939-ANXA5-RNAi).The expression of ANXA5 protein in each group of cells was detected by WB method;the cell migration ability of each group was observed by cell scratch test;the invasive ability of each group was detected by Transwell assay;the cell proliferation was detected by MTT assay and the cell apoptosis was detected by flow cytometry.Results The expression of ANXA5 protein in KD group was significantly decreased(p<:0.05);the migration ability in KD group was significantly decreased(p<0.05);the number of cells in KD group passing through the basement membrane was also significantly reduced(p<0.05);the cell proliferation in KD group was significantly slowed down(p<0.05);and the apoptosis in KD group increased significantly(p<0.05),compared with the other control groups.Conclusion QBC939-ANXA5-shRNA cells can not only decrease the expression of ANXA5 protein,inhibit the migration and invasion of the cells,inhibit the proliferation of QBC939 cells,but also induce the apoptosis of QBC939 cells,which can provide experimental basis for future research.