| ObjectiveHTLV-1-encoding oncoprotein Tax is considered to be the key factor leading to the virus infected host cells malignant transformation.In this study,by constructing a oncoprotein Tax stably expressing cell line named TaxP cells and performing gene expression profile analysis using gene chip,we observed 38 high expressed genes and 12 low expressed genes in Tax P cells,including early growth response factor 1(EGR1).The purpose of this work was to identify the regulation of EGR1 expression in TaxP cells,and further study the expression characteristics and biological mechanism of EGR1.Methods1.EGR1 expression in Tax-positive cells(1)EGR1 expression in HTLV-1-positive T cells HTLV-1 negative T cell lines Jurkat cells and MOLT4 cells,and HTLV-1-positive T cell lines MT2 cells and MT4 cells were chosen.Expression of EGR1 mRNA was detected in those different cell lines by quantitative real-time PCR and the protein expression of EGR1 was determined by using western blot;(2)Effect of Tax on EGR1 expression in T cells The laboratory constructed cell line TaxP that is able to stable expression of Tax and control cell line TaxN were used to test EGR1 mRNA expression by real-time PCR and Tax protein expression via western blot.And the dose-dependent effect of Tax plasmid on expression of EGR1 protein and mRNA was detected in those cells by gene transfection technique.(3)Impact of HTLV-1 infection on expression of EGR1 in host cells HTLV-1-positive T cells MT2 cells were co-cultured with Hela cells or Jurkat cells at a ratio of 1: 5 for 24 h,then the suspended cells(MT2)were removed with medium by repeated washings.EGR1 mRNA expression was determined with real-time PCR and the expression of EGR1 protein was detected by using western blot.2.Characteristics of the expression regulation of EGR1 in Tax-positive cells(1)Construction of different lengths of EGR1 reporter gene and its relative mutants A pair of primers was designed and jurkat cell genome was used as template,the EGR1 fragment(993bp)was amplified with a PCR.After sequencing,the fragment was inserted into the luciferase reporter gene vector Pgl3-basic-luc,which yields Pgl3-EGR1-luc marked E1.Then using Pgl3-EGR1-luc as template,varying degrees of regulatory sequences were amplified by performing PCRs including truncated body E2(-423bp~+1bp),E3(-223bp~+1bp);Based on our preliminary results,Construction of regulatory sequence of NFκB binding site NBS(-422bp~-401bp)deletion and point mutation,marked DelE and MutE respectively,were commissioned by Shanghai Sangon Biotech;(2)Reporter gene activity analysis E1,E2,E3,DelE and MutE were co-transfected respectively with reference vector Prl-luc and p65 shRNA or control vector into Tax P cells and HTLV-1-positive MT2 cells.Cells were harvested after 48 h of transfection.And the luciferase reporter gene activity of EGR1 regulatory regions was calculated;(3)Effect of HTLV-1 infection on reporter gene activity E1,E2,E3,DelE and MutE were co-transfected respectively with reference vector Prl-luc and p65 shRNA or control vector into Hela cells and jurkat cells.Then 1/5 number of MT2 cells were added and cultured for 48 h.The luciferase reporter gene activity of EGR1 regulatory regions was calculated;(4)Direct binding of p65 to EGR1 regulatory region was defined by chromosomal co-precipitation assay A certain number of MT2 cells was collected,Direct binding of p65 to EGR1 regulatory region was defined by chromosomal co-precipitation assay.3.Analysis of p65-mediated EGR1 expression:(1)Impact of NFκB inhibitor BAY 11-7082 on expression of EGR1 5uM BAY 11-7082 or the same volume of control buffer DMSO were added to MT2 cells and Tax P cells.Cells were collected after 24 h for detecting EGR1 and Tax expression using western blot;MT2 cells and TaxP cells were transfected with p65 shRNA or control vector for 24 h.Expressions of p65 protein and EGR1 were estimated by western blot;(2)Effect of different Tax Mutants on expression of EGR1 in host cells Hela cells were transfected with the two classical Tax mutants M22,M47 and the wild-type Tax respectively.Expressions of p65,and EGR1 were determined by western blot;Different lengths of EGR1 luciferase reporter gene was co-transfected with M22,M47 and Tax respectively into 293 T cells for 24 h.The activity of EGR1 regulatory region was detected by promega dual reporter gene assay.293 T cells were co-transfected with NFκB luciferase reporter gene and M22,M47,Tax respectively,the activity of NFκBluciferase reporter gene was detected and plasmid characteristics was verified after 24 h.4.Imploring the impact of EGR1 on RELA expression in HTLV-1-infected cells and its mechanisms Different concentrations of EGR1-expressing plasmid were electroporated into TaxP cells and MT2 cells,expressions of EGR1,p65 and Tax were characterized by western blot;The EGR1-expressing plasmid or control vector were transfected in TaxP cells for 24 h,then nucleoprotein was extracted.Western blot detecting for p65,Lamin B and Tubulin.The influence EGR1 to the nucleus entrance of p65 was observed;Different concentrations of EGR1-expressing plasmid were co-transfected with a luciferase reporter gene pNFκB-luc into TaxP cells and MT2 cells,24 h later,luciferase activity was tested by using promega dual reporter assay.Moreover,nucleoprotein was extracted and the capability of NFκB binding DNA was detected by gel electrophoretic mobility EMSA and the impact of overexpression of NFKB on EGR1 activity was observed.5.Study on the mechanism of EGR1 activating NFκB in HTLV-1-infected cells Jurkat cells were stimulated with PHA or PBS,then the protein synthesis inhibitor cycloheximide(CHX)was added.Proteins were extracted and EGR1 expression was tested by western blot;Jurkat cells were transfected with the Tax-mCherry plasmid for 24 h,then CHX was added.The expressions of EGR1 and Tax were detected by western blot.The effect of Tax on the half-life of EGR1 was observed;the direct interaction between Tax and EGR1 in Tax P cells was determined via co-immunoprecipitation.The mechanism of Tax extending the half-life of EGR1 was studied.6.Analysis of the replicative dynamics of HTLV-1 in Hela cells HTLV-1-positive T cells MT2 cells were added to adherent Hela cells at a ratio of 1.The suspended cells were removed after 1h by repeated pipetting.The cells were collected after 0h,4h,8h,16 h,24h of culturing;expression of HTLV-1 virus core antigen p19 was assessed with western blot.The viral nucleic acid copy number was detected using real-time PCR and the reproduce dynamics of HTLV-1 in Hela cells was estimated.7.Evaluating the mechanism of HTLV-1 infection regulated by EGR1(1)Effect of HTLV-1 infection on host cell apoptosis HTLV-1-positive T cells MT2 cells were added to adherent Hela cells at a ratio of 1.The suspended cells were removed after 1h by repeated pipetting.Cas3/9 activity were tested after Hela cells infected with virus by promega Caspase3/7 Glo and Caspase 9 assay,or proteins were collected and the activity of Cas3/cas9 was checked by western blot.(2)Impact of HTLV-1 infection on host cell autophagy HTLV-1-positive T cells MT2 cells were added to adherent of Hela cells at a ratio of 1.LC3 B expression was observed using western blot and the host cell autophagy was evaluated after virus infection;Hela cells were transfected with plasmid LC3B-EGFP,then HTLV-1-positive T cells MT2 cells were added to the adherent Hela cells at a ratio of 1.The suspended cells were removed after 1h by repeated pipetting,the number of autophagy was measured by confocal microscopy.(3)Influence of interfering EGR1 on the replicative kinetics of HTLV-1 Hela cells were transfected with EGR1 siRNA for 24 h,then an equal amount of MT2 cells were added and co-cultured for another 1h,MT2 cells were removed by repeated pipetting.24 h after MT2 cells added,Cas3/9 activity were measured after Hela cells infected with virus by promega Caspase3/7 Glo and Caspase 9 assay,Cas3/cas9,LC3 B and p19 were detected via western blot respectively,the viral nucleic acid copy was assessed by real-time PCR.Results1.EGR1 expression in Tax-positive cells(1)EGR1 expression in HTLV-1-positive T cells HTLV-1 negative T cell line Jurkat cells,MOLT4 cells,HTLV-1-positive T cell line MT2 cells and MT4 cells were used in this study.We observed that the expressions of EGR1 mRNA and protein in HTLV-1-positive T cell line are at least three times higher than in the control cell line,showing significant difference(P <0.01).The result elucidates that HTLV-1 has the capability to enhance EGR1 expression in host cells;(2)Effect of Tax on EGR 1 expression within T cells The real-time PCR data shows that the amount of EGR1 mRNA in Tax P cells expressing Tax stably is nearly three times higher than in the control cell line TaxN cells.We obtained that the more expression vector of Tax transfected,the more EGR1 mRNA expressed within cells.The significant differences were observed(P <0.01);Western blot data indicated that either Tax stable expression or transient expression,the expression of EGR1 will be significantly facilitated(P <0.01),also the amount of plasmid transfected and the expression level of EGR1 displayed a positive correlation.This study reveal that oncoprotein Tax encoded by HTLV-1 is able to induce up-regulated expression of EGR1 in host cells;(3)Effect of HTLV-1 infection on EGR1 expression in host cells When the co-cultured Hela cells or Jurkat cells were detected,we observed that increasing MT2 cells proportion in the co-culture system resulted in enhanced expression of Tax encoded by HTLV-1.Moreover,the expressions of EGR1 mRNA and protein were also significantly increased.The data suggests that the early HTLV-1 infection could lead to the increased EGR1 expression in host cells.2.Characteristics of expression regulation of EGR1 in Tax-positive cells(1)Construction of different lengths of EGR1 reporter gene and its mutants The full-length luciferase reporter gene E1 of EGR1 regulatory sequences as well as varying degrees of truncations E2(-423bp~+ 1bp),E3(-223bp~+ 1bp)and NFκB binding site NBS(-422bp~-401bp)-mutated DelE and-413/T-G point mutation MutE were constructed successfully.(2)Analysis of reporter gene activity Transfection of luciferase reporter gene EGR1 into TaxP cells and MT2 cells respectively or co-transfected with p65 shRNA,the luciferase activities of E1 and E2 were observed significant higher than that of E3,DelE and MutE,showing that the expression regulation area of EGR1 locates mainly in NFκB binding site(-422~-401bp),which suggesting that p65 is involved in EGR1 expression in Tax positive cells;(3)Impact of HTLV-1 infection on reporter gene activity E1,E2,E3,DelE and MutE were co-transfected respectively with reference vector Prl-luc and p65 shRNA(or control vector)into Jurkat cells.We obtained that the luciferase activities of both E1 and E2 had no significant differences with or without HTLV-1 infection;after silencing p65 with shRNA,no significant differences were observed,showing that the expression of EGR1 is regulated by p65 in the early stage of HTLV-1 infection;(4)Characteristics of the interaction between p65 and EGR1 regulatory regions by chromosomal co-precipitation assay The chromosomal co-precipitation assay data shows that p65 is capable of direct binding the regulatory regions of EGR1,and the binding site locates at-505bp~-223 bp.3.Analysis of p65-mediated EGR1 expression(1)Impact of NFκB inhibitor BAY 11-7082 on EGR1 expression As well as silencing p65 with shRNA,the expressional level of EGR1 was decreased to 50%.Similarly when NFκB inhibitor BAY 11-7082 was used,the expression of EGR1 was down-regulated nearly 60%;(2)Effects of different Tax mutants on EGR1 expression in host cells Tax mutants M22,M47 and wild-type Tax were transfected into Hela cells.The EGR1 expression was significantly higher in the group co-transfected mutant M47 with wild-type Tax as compared to the M22 group.When the different lengths of EGR1 luciferase reporter genes were co-transfected with M22 and M47 respectively into 293 T cells,we found that the luciferase activities of E1 and E2 were dramatically higher in the group transfected with wild-type Tax and M47 than in M22 group(P <0.05),while the activities of E1 and E2 were no notable difference with E3,DelE and MutE activities in M22-transfected group(P> 0.05).Collectively,this work elucidates that M22 mutant that is unable to activate NFκB pathway lost strongly the capability of enhancing EGR1 expression.4.Effect of EGR1 on RELA expression in HTLV-1 infected cells and the mechanisms study Different concentrations of EGR1 were electroporated into Tax P cells and MT2 cells.We found that EGR1 could increase the expression of p65 and NFκB activity.This effect became more significant when increasing dose of EGR1 were used.Also,EGR1 was able to induce p65 entering into the nucleus;EMSA analysis showed that EGR1 could promote the interaction of NFκB with DNA.Taken together,these data show that EGR1 contributes to increase NFκB activity in Tax-positive cells.Combining with our previous result that EGR1 is positive regulated by NFκB,suggesting there is a positive feedback regulation between EGR1 and NFκB in Tax positive cells.5.Investigation of the mechanisms for EGR1 promoting NFκB activity in HTLV-1 infected cells By using the protein synthesis inhibitor CHX,we discovered that the half-life of EGR1 was greatly prolonged in the presence of Tax.And Tax could interact directly with EGR1 from the co-immunoprecipitation assay.It suggests that the increase of the half-life of EGR1 by HTLV-1 might be in a Tax-dependent manner,which leads to EGR1 accumulation and causes further the constant activation of the positive feedback between EGR1 and NFκB in host cells.6.Analysis of HTLV-1 replication dynamics in Hela cells The HTLV-1-positive T cells MT2 cells were added to adherent cultured Hela cells at a ratio of 1.The western blot data show that the expression of HTLV-1 core protein p19 was detectable after 4h and decreased after 8h,then increased gradually;the real-time PCR result demonstrates that the viral nucleic acid copies increases gradually with time.7.Analysis of the mechanisms of HTLV-1 infection regulated by EGR1(1)Effect of HTLV-1 infection on host cell apoptosis The promega Glo assay data display that,after infecting Hela cells with HTLV-1,the activities of cas3/7 and cas9 were no significant difference(P> 0.05)as compared to that in 0h group respectively.Furthermore,there were no significant differences for activated cas3/9 at different times with western blot detection,indicating that HTLV-1 infection does not induce cell apoptosis;(2)Impact of HTLV-1 infection on host cell autophagy LC3 B expressions in groups in which Hela cells were infected with HTLV-1 were notably higher than that in non-infected group(0h),the LC3 B expression was gradually increased over time(P <0.05),The confocal microscopy analysis showed that the number of autophagosome in HTLV-1-infected Hela cells was much higher than that in non-infected group,and the number goes up with times,explaining that HTLV-1 infection could induce autophagy of host cells and this induction is enhanced over time.(3)Influence of interfering EGR1 on the replicative kinetics of HTLV-1 Silencing EGR1 by siRNA in host cells,and there were no significant difference for Cas3/9 activity and expression observed between siRNA and the control groups.However,the autophagy protein LC3 B and HTLV-1 core protein p19 were significantly reduced and the number of autophagosome was also decreased in siRNA group as compared to the control group.The work suggests EGR1,by regulating autophagy rather than apoptosis,is involved in the replication kinetics of HTLV-1 in host cells.Conclusions1.The high expression of EGR1 in HTLV-1-infected host cells is regulated by NFκB.2.There is a positive feedback regulation mechanism between EGR1 and NFκB in Tax-positive cells,the oncoprotein Tax is enable to bind directly EGR1 and prolong its half-life,which might be the key factor of the positive feedback loop between EGR1 and NFκB.3.HTLV-1 infection could induce the host cell autophagy,the impaction of EGR1 on viral replication depends upon regulating the autophagy of HTLV-1 infected cells. |
PDF Full Text Request