The Study Of The Effect Of NLS-RARα Gene On The Proliferation Of HL-60Cell And The Differentiation Of HL-60Cell Induced By ATRA And Relevant Mechanism

PARTⅠ Construction of Recombinant Adenovirus carryingNLS-RARα Gene and Its Expression in K562CellObjective: To construct recombinant adenovirus carrying NLS-RARαgene and detect its expression in k562cells.Methods: The NLS-RARα gene was cloned with the template ofplasmid of PGBKT7-PML-RARα by PCR,and sub cloned into vectorpAdTrace-TO4.The recombinant vector pAdTrace-TO4-NLS-RARα wasdigested with PmeI, and transformed into competent BJ5183bacteriawhere the plasmid was recombined with pAdEasy-1by homologousrecombination forming recombinant adenovirus plasmidpAd-NLS-RARα. The recombinants which were digested with PacⅠ weretransfected into AD293cells for packaging and then obtain recombinantadenovirus Ad-NLS-RARα. After amplified for four times，the adenovirustiters were tested. AD293cells were infected by adenovirus, and identifiedthe expression of NLS-RARα gene in K562cell by RT-PCR and western-blot.Results: The recombinant adenovirus plasmid Ad-NLS-RARα wasconstructed successfully. The adenovirus titers were up to7.2×108pfu/ml.The specific expression of NLS-RARα gene was identified by RT-PCR andWestern-blot in K562cell.Conclusion: High titer recombined adenovirus carrying NLS-RARαgene was successfully obtained. K562cell which infected by recombinedadenovirus Ad-NLS-RARα highly expressed NLS-RARα gene. PART Ⅱ Effect of Recombinant Adenovirus carryingNLS-RARα Gene on the Proliferation of HL-60Cell and theDifferentiation of HL-60Cell Induced by ATRA and RelevantMechanismObjective: To explore the effect of recombined adenovirus carryingNLS-RARα gene on proliferation of HL-60cells and the differentiation ofHL-60cells induced by ATRA.Methods: HL-60cells was infected with Ad-NLS-RARα and controlvirus Ad-KZ. The efficiency of infection was detected by FCM. The mRNAand protein levels of NLS-RARαwere assessed by Real-time PCR (RT-PCR)and Western blot, respectively. MTT assay were applied todetermine proliferation of HL-60cells. Cell surface differentiation antigenCD11b of infected HL-60cell induced by ATRA was examined by FCM.The mRNA and protein levels of C-MYC of infected HL-60cell induced byATRA were determined by Real-time PCR (RT-PCR) and Western blotassay.Results: The efficiency of infection of Ad-NLS-RARαand Ad-KZ onHL-60cell was70%-80%.The mRNA and protein levels of NLS-RARαgeneof HL-60cells which infected by Ad-NLS-RARαwere both obviouslyhigher than the cells which infected by Ad-KZ and non-infected(P<0.05).The proliferation ability of HL-60cell infected by Ad-NLS-RARαwassignificantly increased (P<0.05).The level of CD11b of HL-60cell infectedby Ad-NLS-RARαand induced by ATRA was clearly decreased than controlgroups(P<0.05). The mRNA and protein levels of C-MYC gene of HL-60cells infected by Ad-NLS-RARαand induced by ATRA were both obviouslyhigher than the cells which infected by Ad-KZ and non-infected(P<0.05).Conclusion: The recombined adenovirus Ad-NLS-RARαcan increasethe proliferation ability of HL-60cell, and inhibit the differentiation ofHL-60cell through reduce the expression level of C-MYC gene. PART Ⅲ Effect Of RNA Interference Targeting NLS-RARαGnen On Proliferation And Differentiation Of HL-60CellsObjective: To explore the effect of NLS-RARα gene expressioninhibition on proliferation and differentiation of HL-60cells.Methods: The shRNA eukaryotic expressing vector targetingNLS-RARαwas constructed and was transfected into HL-60cells byLipofectamineTM2000liposome. The mRNA and protein levels ofNLS-RARαwere assessed by Real-time PCR (RT-PCR)and Western blot,respectively. MTT assay were applied to determine proliferation of HL-60cells. Cell cycle and cell surface differentiation antigen CD11b were ofinterference group cells were both obviously down regulated (P<0.05); Theproliferation ability measured by flow cytometry (FCM).Results: The mRNA and protein expression of NLS-RARαofinterference group cells was reduced (P<0.05); FCM analysis indicated thatthe number of interference group cells in G1/G2phase was obviouslyincreased, meanwhile the number of cells in S phase was clearly lower thancontrol groups, and the expression of CD11b was also increased(P<0.05). Conclusion: The inhibition of NLS-RARαexpression can efficientlysuppress cell proliferation and promote cell differentiation..