Effect Of PML(NLS~-) Gene On Proliferation And Apoptosis Of HL-60Cells

PARTⅠ Construction and identification of RecombinantAdenovirus Ad-PML（NLS~-）Objective To construct recombinant adenovirus expressing PML（NLS~-）protein and observe its’ effect on the proliferation of k562cells.MethodsThe PML（NLS~-）gene was cloned with the template of plasmid ofpCMV-HA-PML （NLS~-） by PCR,and inserted into vectorpAdTrace-TO4.The recombinant vector pAdTrace-TO4-PML（NLS~-）wasdigested with PmeⅠ,and transformed into competent BJ5183bacteriaswhere the plasmid was recombined with pAdEasy-1by homologousrecombination forming recombinant adenovirus plasmid pAd-PML（NLS~-）.The recombinants which were digested with PacⅠweretransfected into AD293cells for packaging,acquiring recombinantadenovirus Ad-PML（NLS~-）.After amplified of four times,the adenovirustiters were tested.AD293cells were infected by adenovirus,and identifiedthe expression of PML（NLS~-）gene by RT-PCR and western blotting.Theproliferation of K562cells was tested with MTT method.Results Therecombinant adenovirus plasmid pAd-PML （NLS~-）was constructed successfully via restricted digestion、PCR and sequencing. The adenovirustiters were up to1x1010ｐｆｕ／ｍｌ.Both of RT-PCR and westernblotting results revealed that the PML（NLS~-）gene cloned into Ad-PML（NLS~-）could express in AD293cells.MTT assay proved that theproliferation of K562cells infected by Ad-PML（NLS~-）was enhancedsignificantly compared with Ad-KZ group and non-infectedgroup.Conclusion The recombinant adenovirus Ad-PML （NLS~-）waspackaged and amplified，and the Ad-PML（NLS~-） group could enhanceproliferation of K562cells. PARTⅡ Effect of PML(NLS~-) gene mediated by recobinantadenovirus vector on apoptosis of HL-60cells andmechanismObjective To investigate the effect of PML(NLS~-) gene on theemodin-induced apoptosis of human HL-60cells and mechanism.MethodsHL-60cells were infected by recombinant adenovirus Ad-PML（NLS~-）andAd-KZ. PML（NLS~-）gene was detected by Real-time PCR and Westernblotting. The proliferation level of HL-60cells was determined by MTTmethod. HL-60cells were treated with80μmol／L emodin for72h，and then analyzed for cell cycle and apoptosis rate by flow cytometry， fortranscription levels of apoptosis-related BCL-2、BAX and C-MYC gene byRT-PCR，and for translation levels of the genes by Western blot．ResultsCompared to normal control group and Ad-KZ group, the mRNA andprotein expression level of PML（NLS~-）gene of HL-60cells infected byAd-PML（NLS~-）were arised significantly by RT-PCR and Western blot. Theproliferation level of HL-60cells infected by increased significantly.Thepercentage of HL-60cells treated with80μmol／L emodin at G1phasedecreased and at S phase increased,while the emodin-induced apoptosis wasrelived.Meanwhile，the mRNA transcription and protein expression levels ofBax gene decreased significantly,while those of BCL-2and C-MYC genesincreased significantly.Conclusion The over-expression of PML（NLS~-）gene might promoted the proliferation and arrest the apoptosis of HL-60cells by up-regulating the expressions of BCL-2and C-MYC genes anddown-regulating the expression of BAX gene． PART Ш Effect Of RNA Interference Targeting PML(NLS~-)on Proliferation and Apoptosis of HL-60cellsObjective To investigate the effect of RNA Interference TargetingPML(NLS~-) on proliferation and apoptosis of HL-60cells. Methods ShRNA sequence targeting PML(NLS~-) was designed,which was clonedinto the vector to form the recombinant vector. HL-60cells weretransfected with recombinant plasmids and negative control in mediationof Lipofectamine Reagent.The target gene was detected by Real-timePCR and Western blotting. MTT method assayed the proliferation rate ofHL-60cells.FCM assayed cell cycle and cell apoptosis level. Results ThemRNA and protein expression of PML(NLS~-) of interference group cellswas reduced; MTT results showed that the proliferation level ofinterference group cells was decreased(P<0.05),and the difference of thesecond day was significant(P<0.01)；Cell cycle results showed that theinhibition PML(NLS~-) gene’s expression could increase the ratio of HL-60cells’S phase,and decrease the ratio of G1and G2phase；AnnexinV/7-AAD results showed that the rate of apoptosis of the interferencegroup was significantly higher than negative control group.ConclusionThe inhibition of PML(NLS~-) gene expression could suppress theproliferation and promote the apoptosis of HL-60cells.