Research Of Lentivirus-mediated PLD₂ Gene Knock-down In HL-60 Cells

Objective:Konckdown the PLD₂ gene expression of human promyelocytic leukemia cell line HL-60 by RNAi specifically. This will lays a foundation for further study of the biological function of PLD2 and the treatment of leukemia in clinical.Methods:1. shRNA was desigened by Invitrogen's online RNAi Designer.2. PLD2 RNAi lentivirus vectors were constructed with BLOCK-iT^TM Lentivirual Polâ…¡miR RNAi Expression System from Invitrogen.3. 4 plasmides was co-transfected into the 293FT cells mediated by lipofectamin 2000 so as to produce the lentivirus.4. Infected the 293FT cells by the lentivirus and determine the titre by flow cytomerty after infection.5. HL-60 cells were infected with the lentivirus and the expression of PLD₂ mRNA and protein were analyzed by RT-PCR and Western Blotting, respectively.6. The kinetics of cell proliferation was determinated by MTT assay.Results:1. pcDNA^TM6.2-GW/EmGFP-PLD₂ miR Vectors were successfully constructed.2. Successfully constructed pLenti6/V5-GW/EmGFP-PLD₂-miR Vectors. After co-transfected it into 293FT cells with 3 other plasmids, Lentiviruses were harvested from transfected 293FT cells.3. In lentivirus-transfected HL-60 cells, the expression of PLD₂ mRNA and protein were found to be significant lower than that in the untransfected control group accordingly.4. Significant higher apoptotic rate of HL-60 cells was observed in PLD2 RNA-interfered group.5. MTT assay showed that various concentrations of rhPLD₂ inhibit the growth of HL-60 cells in a dose-dependent manner.conclusions:The PLD₂ gene was successfully silenced in HL-60 cells by RNAi, leading to the low expression of PLD₂ and the increased cell apoptosis. This result suggests the inhibitory effect of PLD₂ expression on the apoptosis of HL-60 cells and the possibility of PLD₂ gene knockdown as a new strategy in the clinical treatment of leukemia.