Effect Of HOXA10Gene Silence Mediated By Lentivirus On The Proliferation,Apoptosis And Drug Resistance Of U937Cells In Vitro And In Vivo

Objective:To study the impact of HOXA10gene silence on the proliferation and drug sensitivity of leukemia cell lines in vitro and in vivo.Methods:Fristly, U937cells were transfected by Lipofectamine2000with plasmid, adenovirus vector and lentivirus vector respectively.And their results were evaluated by the means of transfection efficiencies and cell viability.Secondly, Four different shRNA expression plasmid vectors were built to interfere with HOXA10gene and as well as its overexpression vector. The RNAi efficiency of the four interference plasmid vectors was determined by Western blot. The best one was chosen to packaged lentivirus(lenti-shHOXA10).U937cells’HOXA10gene expression at mRNA and protein level was detected by Real-Time PCR and Western Blot.U937cells were divided into interference group,negative control group and blank control group. Cell morphology changes were observed by Wright staining.Cell survival was determined by MTT assay and apoptosis rates were detected by flow cytometry.Thirdly,Targeted down-regulation of HOXA10gene expression,the sensitivity of U937cells to Ara-c,DNR and VCR was determined by MTT assay and apoptosis rates were detected by flow cytometry.Fourthly, The NC and KD group’s GFP-positive cells were sorted by flow cytometry and then U937xenografts were established by subcutaneous injection of U937cells (viability>90%) into the flanks of nude mice.Tumor inhibition rate of KD,NC+VCR and KD+VCR groups were calculated.Results:Fristly, Both fluorescence microscopy and flow cytometry results showed that GFP positive cell rates were low in Lipofectamine2000and adenovirus groups. Transfected by lentivirus, the field of vision was full of GFP positive cells under fluorescence microscopy and GFP positive cell rate was about90%.Trypan blue staining showed that the rate of living cells were significantly higher in adenovirus and lentivirus groups than in the Lipofectamine2000group (p<0.01).Secondly, Lentiviral-shRNA vector of HOXA10gene was successfully constructed The expression of HOXA10gene of U937cells at mRNA and protein level was decreased by about92.3%and91.14%,respectively.The proliferation inhibition rate it’s about (43.9±0.7)%and the apoptosis rates it’s about (27.1±1.4)%in KD group;and the differences between the KD and control group (NC、BC) was significant (p<0.05).Wright’s staining showed that the ratio of karyon to cytoplasm is reduced and mitotic phase is rare in KD group.Thirdly,MTT and FCM results showed that:down-regulation of HOXA10expression levels can reduce the IC50and improve the apoptosis rates of Ara-c, DNR, VCR to U937cells;compared with the control group (NC, BC),the differences was significant (p<0.05).Fourthly, xenograft AML model in nude mice was successfully established; compared with the NC group,tumor inhibition rates were26.55%in KD group,39.86%in the NC+VCR group and87.87%in KD+VCR group,which show that the effect of RNA interference and VCR was better then their working alone; RNA interference and VCR could synergistically work to inhibit the growth of U937cells in nude mice.Couclusions:Fristly, Lentivirus was an ideal vehicle for U937cell transfection. Secondly, Lentivirus mediated RNAi,which can steadily reduce the expression level of HOXA10, can effectively inhibit the proliferation and promote apoptosis of U937cells. Thirdly, down-regulation of HOXA10gene can enhance the sensitivity of U937cells to chemotherapeutic drugs,such as cytarabine, daunorubicin and Vincristine. Fourthly, Lentivirus vector-mediated RNA interference targeted HOXA10gene could enhance the sensitivity of U937cells to VCR in vivo.