The Expression Of CD70 In Hepatocellular Carcinoma And Its Suppressive Impact On Proliferation Of Lymphocytes

Objective:To investigate the expression of CD70 gene in hepatocellular caicinoma and its role in the immune escape of hepatocellular carcinoma.Method:1.The expression of CD70 mRNA in hepatocellular carcinoma and its clone and expression(1)Detect the expression of CD70 mRNA in hepatocellular carcinoma tissue and hepatocellular carcinoma cell lines using RT-PCRSpecific primer for the CD70 gene was designed.Extract the total RNA of hapatocellular carcinoma tissue and hepatocellular carcinoma cell lines then RT-PCR to anallyze the expression of CD70 mRNA.(2)Construct CD70 expression vector and study its expression in vitroSpecific primer for the CD70 gene were designed.Then human CD70 was amplifie by RT-PCR and cloned into the vector pcDNA3.0 to construct the expression plasmid pcDNA3-CD70.The recombinant was transformed into JM109,the positive clone was identified by restrictive enzymeesassay,PCR,automatic DNA sequencing repectively.In order to assay the expression of CD70 in vitro,pcDNA-CD70 was transfected into HepG2 cells.24 hours after transfection,the expression of CD70 was determined by RT-PCR and Western blot.(3)Construct CD70 high expression vector and study its expression in vitroSpecific primer for the CD70 gene were designed.Then human CD70 was amplified by RT-PCR and cloned into the vector PRK to construct the expression plasmid PRK-CD70.The recombinant was transformed into JM109,the positive clone was identified by restrictive enzymeesassay,PCR,automatic DNA sequencing repectively.In order to assay the expression of CD70 in vitro,PRK-CD70 was transfeted into HepG2 cells.24 hours after transfection,the expression of CD70 was determined by RT-PCR and Western blot.2.The suppression of CD70 on proliferation of lymphoeytes in human hepatocellular carcinoma cell lines(1)CD70 function of inhibiting lymphocytes proliferation was assessed by Cell counting kit 8(CCK8)CD70 eukaryotic expression vector(CD70-pcDNA3,CD70-PRK)was constructed and transiently transfected into HepG2 cell line.24 hours after transfection,coculture HepG2 with Jurkat for 24 hours,CD70 function of inhibiting lymphocytes proliferation was assessed by Cell counting kit 8(CCK8).(2)Detect the expression of CD27,Siva mRNA in Jurkat after activation using RT-PCRExtract the total RNA of jurkat cells and activated jurkat ceils then RT-PCR to analyze the expression of CD27,Siva mRNA.Results1.the expression of CD70 mRNA in hepatocellular carcinoma tissue and hepatocellular carcinoma cell linesCD70 gene is present in BEL-7402 and SMMC-7721 but not in HepG2.There are3 cases that have the expression of CD70 in 10 cases of hepatocellular carcinoma while CD70 is not expressed in the liver tissue adjacent to cancer tissue(2 cases).2.CD70 expression vector was successfully constructed and its expression in HepG2 cell line was assayedEnzymes digestion,PCR and automatic DNA sequencing confirmed that the recombinant vectors pcDNA3-CD70 and PRK-CD70 were successfully constructed. These two plasmids were transfected into HepG2 cell line to assay gene expression. 24 hours after transfection,CD70highly expressed in RNA and protein level which was assayed by RT-PCR and Western blot.3.The expression of CD70 in HepG2 cell line can inhibit the proliferation of jurkat cells CD70 eukaryotic expression vector(CD70-pcDNA3,CD70-PRK)were successfully constructed.and transiently transfected into HepG2 cell line.The HepG2 cell transfected with CD70 gene can significantly inhibit the proliferation of PHA-activated Jurkat cell.The proliferation inhibition rate of Jurkat cell cocultrued with HepG 2 cell transfected by CD70 gene is 65.03%,80.36%respectively,which is significan-tly higher than that of Jurkat cells cocultrued with HepG2 cell controls.4.The expression of CD27,Siva mRNA in Jurkat after activation assayed byRT -PCRRT-PCR results showed that the expression of CD27,Siva mRNA in PHA-activated Jurkat cell were higher than that of jurkat cell without PHA activation.Conclusion:This study firstly detect the expression of CD70 in hepatocellular carcinoma tissue and hepatocellular carcinoma cell lines using RT-PCR.RT-PCR results showed that CD70 gene is present in BEL-7402 and SMMC-7721 but not in HepG2.There are 3 cases that have the expression of CD70 in 10 cases of hepatocellular carcinoma while CD70 is not expressed in the liver tissue adjacent to cancer tissue(2 cases).This study successfully constructed eukaryotic expression vector (pcDNA3-CD70)and highly-expressed eukaryotic expression vector(PRK-CD70). The expression of them could be detected in RNA and protein level.These two plasmids were transfected into HepG2 cell line.24 hours after transfection,,coculture HepG2 with Jurkat for 24 hours,the HepG2 cell transfected with CD70 gene can significantly inhibit the proliferation of PHA-activated Jurkat cell.CD70-CD27 can induce apoptosis of lymphocytes.CD27 and fas are both a number of the tumor necrosis facter receptor(TNFR)superfamily.Unlike fas,the cytoplasmic tail of CD27 lacks a death domin.Apoptosis can be induced in the presence of the proapoptotic protein Siva.Siva has a death-domain like region that binds to the CD27 cytoplasmic tail and transmits an apoptotic signal.The expression of CD27,Siva mRNA in Jurkat after activation assayed byRT -PCR.RT-PCR results showed that the expression of CD27,Siva mRNA in PHA-activated Jurkat cell were higher than that of jurkat cell without PHA activation.Thus,we can conclude that apoptosis may be induced in the cells surpressed by CD70.