Expression Of DcR3 And Its Function On The Growth, Migrating And Apoptosis In Hepatocellular Carcinoma

ObjectiveDecoy receptor 3(DcR3),a newly identified member of the tumor necrosis factor receptor(TNFR)super-family,is over-expressed in many various kinds of malignant tumors.The objective of the research is to investigate the expression of DcR3 mRNA and protein in hepatocellular carcinoma(HCC)tissues and HCC cell lines and their correlations with clinicopathologic features,and to elucidate its role in tumorigenesis,development,recrudescence and metastasis, as well as to explore the relationship between the expression of DcR3 and the apoptosis in HCC.Then to observe the change of the biological characteristics(growth and proliferation,movement and transference,apoptosis,sensitivity to CDDP)in the hepatocellular carcinoma cell line with positive DcR3 expression adding the neutralization antibody DcR3.Materials and Methods①The expression of DcR3 mRNA was detected by semi-quantitive reverse transcription-polymerase chain reaction(RT-PCR)in 69 cases of HCC and the adjacent-tumor liver tissues.The relationship between the relative content of DcR3 mRNA and clinicopathological features of HCC was analyzed.②The tissue microarrays comprised 125 cases of HCC tissues, 48 adjacent-tumor liver tissues,64 liver cirrhosis tissues and 25 normal liver tissues,Immunohistochemistry(EnVision method)was employed to detect the expression of DcR3 proteins.Statistic analysis was made to figure out the correlation between the expression and the clinicopathological features of HCC③The mRNA and protein expression of DcR3 were detected in 39 cases of HCC and their para-tumor tissues by RT-PCR and immunohistochemistry,respectively.The apoptosis was evaluated by the method of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL)and DNA agarose electrophoresis.Statistic analysis was made to figure out the correlation among the DcR3 expression,apoptosis and the clinicopathological features of HCC.④Human hepatocellular carcinoma cell lines:HepG2,HepB3, SMMC-7721,bei-7402,bel-7405:liver cell line of para- tumor tissues QSG-7701 and normal liver cell line HL-7702 were harvested without any stimulation.The mRNA expression of DcR3 was detected by RT-PCR and the expression of DcR3 protein was detected by immunocytochemistry.The neutralization antibody DcR3 was added into different liver cancer cell lines and the cell livability rate was detected to decide the cell line for the following experiments and also to search the best working time and concentration of the neutralization antibody DcR3 by MTT assay.The difference of the DcR3 protein expression in hepatocellular carcinoma cell line was checked by immunocytochemistry after adding the neutralization antibody DcR3.The cellular proliferation was examined by cell growth curve and colony formation assay;The changes of cell cycle of cells were analyzed by flow cytometry(FCM);The apoptosis was explored by FCM, DNA ladder,TUNEL and electron microscope;The transferring ability was investigated by the wound healing test;And the sensitivity to Cisplatin(CDDP)of the hepatocellular carcinoma cell line was detected by MTT assay with neutralization antibody DcR3.Results①In 69 cases of HCC tissues,426 bp bands were detected in 67 cases,the positive rate of DcR3 mRNA expression was 97.10%,which is significantly higher than that in adjacent-tumor liver tissues 76.81%(53/69,P＜0.01).The relative content of DcR3 mRNA in HCC was significantly higher than that in the adjacent-tumor liver tissues (P＜0.01).Sequencing analysis showed 4 different point mutations in the DcR3 gene mRNA of HCC in two cases of positive amplification products.The expression was associated with metastasis and the tumor size but had no correlation with age,gender,differentiation grades,clinical stages,cirrhosis,AFP,portal vein tumor embolus,capsular infiltration,tumor nodes or HBsAg.②There were 253 cases that could be used in the tissue microarrays(utilization rate was 96.56%,253/262);The positive rate of DcR3 in HCC tissues was 73.33%(88/120),significantly higher than that in the adjacent-tumor liver tissues 54.17%(26/48, P=0.017),cirrhosis tissues 37.10%(23/62,P=0.000)and the normal liver tissues 8.70%(2/23,P=0.000).The positive rate of DcR3 in adjacent-tumor tissues and cirrhosis tissues were both significantly higher than that in the normal liver group(P=0.000,P=0.005); The positive rate of DcR3 in HCC tissues in the clinical TNMstageⅠ,Ⅱwas 61.76%(42/68),obviously lower than that in the stageⅢ,Ⅳ88.46%(46/52,P=0.001);The positive rate of DcR3 in the cases without metastasis within 20 months was 58.82%(20/34), significantly lower than that in the group with metastasis 96.67% (29/30,P=0.000);The positive rates DcR3 in the groups of AFP≥400μg/L 80.82%(59/73),portal vein tumor embolus 91.30% (42/46),capsular infiltration 84.34%(70/83)and multiple tumor nodes 89.13%(41/46)were significantly higher than that in the groups of AFP＜400μg/L 61.70%(29/47,P=0.021),without tumor embolus 62.16% (46/74,P=0.000),with no capsular infiltration 48.65%(8/37, P=0.000)and single node 63.51%(47/74,P=0.002).It was not associated with patients' age,sex,histological classification, cirrhosis or tumor size.③In 39 cases of HCC tissues,the apoptosis index(AI)and the positive rate of DNAladder in HCC were both significantly lower than that in the non-cancerous group(P=0.000).The AI was related to metastasis,clinical TNM stages and capsular infiltration but had no correlation with age,gender,histological differentiation grades,cirrhosis,plasma AFP levels,portal vein tumor embolus, tumor sizes,number of the tumor nodes or HBsAg.The relative analysis showed that there were negative correlations between AI and the expression of DcR3 mRNA and protein in HCC(r=-0.34,P=0.002; r=-0.679,P=0.000).There was positive correlation between the positive rate of DNA ladder and the expression of DcR3 mRNA and protein in HCC(r=0.62,P=0.000).The expression of DcR3 mRNA was positively related to the expression of protein(r=0.419,P=0.000).④The all of 5 kinds of human hepatocellular carcinoma cell lines expressed DcR3 in mRNA and protein,while QSG-7701 and HL-7702 did not.HepG2 was chosen as the target cell lines for the experiments with the best concentration of neutralization antibody DcR3 0.8mg/L, best time 48h.The DcR3 protein expression became weakening in HepG2 after the DcR3 neutralization antibody DcR3;The cell viability, growing speed and colony formation ability were decreased obviously by adding DcR3-Ab(P＜0.01).The progression of the cell cycle was arrested in Gl/S-phase remarkably induced byDcR3-Ab(P＜0.01); The apoptosis was also significantly increased(P＜0.01);The transferring ability was significantly inhibited(P＜0.01)and the livability of the liver cells with CDDP was decreased by DcR3-Ab.Conclusions①DcR3 mRNA and protein are both over-expressed in HCC tissues and cell lines.The over-expression of DcR3 mRNA and protein may play an important role in the process of carcinogenesis,progression, recrudescence and metastasis in HCC;②Point mutation of the DcR3 gene might exist in HCC;③Tissue microarrays techniqueis a feasible,rapid,economic and accurate approach for screening clinical tissue specimens on a large scale;④The over-expression of DcR3 might inhibit the apoptosis of HCC cells;⑤DcR3 neutralization antibody can restrain the growth and movement,accelerate the apoptosis of the HCC cells,meanwhile increase the sensitivity to CDDP.The DcR3 gene plays an important role in origination,progression and metastasis of liver cancer and it is hopeful to be applied in the future clinical therapy to hepatocellular carcinoma.