The Role And Molecular Mechanism Of GINS2in Acute Promyelocytic Leukemia Pathogenesis

PART ⅠTHE EFFECT OF DOWN-REGULATION OF GINS2ONPROLIFERATION AND APOPTOSIS OF HL60CELLSObjective： To investigate the effect and mechanism of smallinterfering RNA targeted GINS2expression on proliferation and apoptosisof HL60.Method：The expression of GINS2complexes comprised in threeleukemia cell lines HL60,U937,THP-1were detected byReversed-Transcript PCR（RT-PCR）.The plasmid specific targeted GINS2expression were stably transfected into HL60cells with high expression ofthis gene by means of lipidosome-mediated method. Then，the mRNA andprotein level were detected by RT-PCR and Western blot respectively.Meanwhile apoptosis and cell proliferation after transfection weremeasured by MTT method and flow cytometry. In addition，the change ofapoptin Bax and Bcl2were disclosed by western blot. Results：Among three leukemia cell lines，the expression of GINS2from low to high was following as：THP-1,U937,HL60. After siRNAplasmid were transfected into HL60cells，both GINS2expression level ofmRNA and protein in interfering group，measured by RT-PCR andWestern blot method， were down-regulated after transfection whencompared with untreated control group and negative control group.Together, MTT and flow cytometry showed that cell growth wassignificantly inhibited. Moreover， the expression lever of Bax wassignificantly increased whereas Bcl2was dramatically decreased inpositive transfected group.Conclusion： The down-regulation of mRNA and protein level ofGINS2complexes might induce apoptosis and inhibit cell growth by waysof up-regulation of Bax and down-regulation of Bcl2in HL60cell lines. PART ⅡDOWN-REGULATE THE EXPRESSION OF GINS2INHIBITS CELL CYCLE REGULATORS IN HL60CELLSObjective：To observe changes of cell cycle after down-regulation of GINS2in HL60cells and investigate related mechanism.Method： The recombinant plasmid carrying siRNA andnon-homologous sequence were stably transfected into HL60cells asexperimental group and negative control group respectively. Moreover，bank control group only treated by lipidosome and HL60cells that acted asuntreated group. Flow cytometry was used to detect the cell cycle ofHL60，3H-TdR incorporation assay and Colony-forming test wereperformed to measure duplication and nucleic acid synthesis in four groupseither. The protein expression of cycle regulatory proteins CDK1andCyclinB1was measured by western blot. Meanwhile，transcription andtranslation levels of ATM，CHK2,P53was detected by RT-PCR and westernblot respectively.Result：48h after transfection,GFP could be observed by Fluorescencemicroscope. Flow cytometry showed that cell growth was significantlyinhibited and G2/M peak was found in interfering group. Compared withother three groups，nucleic acid synthesis and cell proliferation was blockedin experimental group. CDK1，cyclinB1related with cell cycle regulationwere dramatically decreased over time. Together，Both RT-PCR andwestern blot demonstrated that the transcription and translation levels ofATM，CHK2,P53were notably increased.Conclusion：Down-regulation of GINS2can suppress nucleic acidduplication and influence cell cycle process of HL60by means of ATM， CHK2and P53. PART ⅢCONSTRUCTION OF RECOMBINANT ADENOVIRUSVECTOR CONTAINING GINS2GENE AND THE EFFECTOF GINS2ON PROLIFERATION AND APOPTOSIS OFHL60CELLSObjective： To construct and identify an adenovirus vector ofcontaining GINS2gene and detect its expression in HL60cell lines.Method：GINS2Coding sequence was amplified by RT-PCR usingplasmid pcDNA3.1-GINS2as a template，then cloned to pAdTrace-TO4vector. After Enzyme digestion，transformation and DNA sequencing，thereformed BJ5183-sensitive bacteria that contain pAdEasy-1weretransformed with lined vector cut by Pme I enzyme. The recombinantspAdT-GINS2was transfected HEK293cells to produce virus Ad-GINS2then identified by PCR and fluorescence microscope. Meanwhile， HL60cells were infected by Ad-GINS2after virus titer was detected. MTT assayand Colony-forming test were used to detect the effect of cell proliferationin HL60cells. Flow cytometry were performed to measure apoptosis rate of HL60cells. Bax and Bcl2related with apoptosis were detected western.Result： PCR，Enzyme digestion，DNA sequencing showed thatGINS2gene was cloned to shuttle vector successfully.Meanwhile，recombinant adenovirus pellet was obtained. After three cycles ofamplification，GINS2protein expression could be detected by western blotafter recombinant adenovirus pellet was used to infect HL60cells. MTTassay and Colony-forming test showed that over-expression of GINS2could stimulate proliferation of HL60cells. Flow cytometry demonstratedthat apoptosis rate of HL60cells notably decreased. Western blot showedthat Bax decreased，whereas Bcl2increased dramatically.Conclusion：Recombinant adenovirus vector containing GINS2genehas been constructed successfully，and can stimulate the proliferation ofHL60cell lines. All in all，which lay the groundwork for future research themechanism of GINS2involved in the process of Acute promyelocyticleukemia.