Detection And Analysis Of Related Gene Expression In EBV-Transformed Lymphoblasts

Objective: We have been screened four molecules which may play a key role in theprocess of Epstein-Barr virus-induced lymphocyte transformation, namely E2F1, Plk1,BIRC5and PTPN11, by use of cDNA gene chip technology (Agilent human wholegenome microarray technology). We used real-time PCR and Western blot technologyto detect and analyze the gene expression of E2F1, PLK1, BIRC5, PTPN11in bothEBV-transformed lymphoblasts and their matched healthy lymphocytes. Thisexperiment will verify the cDNA microarray results， and provide the experimentalbasis for studying the molecular mechanism how the EBV induces lymphoma.Method: Human lymphocytes were separated from fresh peripheral blood of fivehealth donors. EBV was used to transform lymphocytes in vitro. Total RNA andProtein were extracted from5cases of the normal lymphocytes and transformedlymphoblasts respectively, then real-time PCR and Western blot technology were usedto detect and analyze the gene expression of E2F1, PLK1, BIRC5, PTPN11in bothEBV-transformed lymphoblasts and their matched healthy lymphocytes.Result: EBV-transformed lymphoblastoid cell lines were established successfully. Weobtained5strains transformed lymphoblastoid cells, and detected the expression ofE2F1, PLK1, BIRC5, PTPN11gene in five cases of transformed lymphoblastoid cellsand their matched healthy human lymphocytes.E2F1gene expressed in both two types of cells, compared with normallymphocytes, the mRNA expression in transformed cells was up-regulated3.161times, and the difference was statistically significant (P<0.05); Western blot resultsshowed that, compared with normal lymphocytes, E2F1protein expression wassignificantly increased in transformed lymphoblastoid cells, the difference wasstatistically significant (P<0.05). PLK1gene expressed in both two types of cells, compared with normallymphocytes, the mRNA expression in transformed cells was up-regulated11.329times and the difference was statistically significant (P<0.001); Western blot resultsshowed that, the expression of PLK1protein in transformed cells was up-regulated8.140times than that in normal lymphocytes, and the difference was statisticallysignificant (P<0.001).BIRC5gene expressed in both two types of cells, compared with normallymphocytes, the mRNA expression in transformed cells was up-regulated7.921times and the difference was statistically significant (P<0.001); Western blot resultsshowed that, the expression of PLK1protein in transformed cells was up-regulated2.065times than that in normal lymphocytes, and the difference was statisticallysignificant (P<0.01).PTPN11gene expressed in both two types of cells, compared with normallymphocytes, the mRNA expression in transformed cells was down-regulated4.760times, the difference was statistically significant (P<0.05); However, Western blotresults showed that the difference of PTPN11protein expression between transformedcells and normal lymphocytes was not statistically significant (P>0.05).Conclusion:1. The levels of E2F1, PLK1, BIRC5mRNA transcription and protein expression intransformed cells were up-regulated than those in normal lymphocytes.2. The level of PTPN11mRNA transcription in transformed cells was down-regulatedthan that in normal lymphocytes. However, the difference of PTPN11proteinexpression was not statistically significant between transformed cells and normallymphocytes.