Effects Of Lentivirus Mediated-WWOX Gene Overexpression On Biological Properties Of Jurkat And K562Human Hematopoietic Malignant Cells

Objective:1.To construct a lentivirus mediated-WWOX overexpression systemand verify its validity and infectious efficiency;2.To investigate the effects oflentivirus mediated-WWOX gene overexpression on biological properties of Jurkatand K562leukemia cells;3.To explore the molecular mechanisms underlying thebiological properties changes.Methods:1. PCR technique was performed to obtain our target gene WWOXclones, the cDNA strands obtained in this process were digested by Age I enzyme. Atthe same time, pGC-FU vector was treated as in the same manner by Age I enzyme tomake it linear enough. The linear WWOX cDNA strands and the linear vectors werebound together with the help of T4DNA ligase and followed by transformation,coated onto agar plate, picked clones and amplification, respectively. The combinedplasmids were extracted and sent to sequencing. After sequencing, the lentivirusvectors combined full length WWOX cDNA strands were propagated and harvestedafter centrifugation, then pGC-FU-WWOX-GFP and pGC-FU-GFP overexpressionsystem was completed. qPCR and Western blot were employed in the followingevaluation tests.2.The effects of WWOX gene overexpression on biologicalproperties of Jurkat and K562cell lines were estimated by cell counting kit8(CCK-8),Clone forming assay, DAPI stain, DNA ladder electrophoresis Annexin V PE/7-AADfluorescence test and Western blot, respectively.3. Indirect immunofluorescence wasemployed to observe the expression and location of apoptosis related proteins such asp53, p73, NF-κB p65and cytochrome C in Jurkat and K562cells. The expressionlevels of apoptosis related proteins including Bcl-2, Bax, cytochrome C, Caspase-9,Caspase-3, p53and p73were identified by qPCR and Western blot after transfection.Immunoprecipitation and Western blot were used to analyze the mutual effects between WWOX and p73, p53.Results:1. The sequencing of the reconstruct plasmids were completely correct,and the virus titer measured via qPCR was2×108TU/ml.2. Stable green fluorescencewas observed after infected for48hours in both two kinds of cell lines. The WWOXexpression level of the overexpresion groups increased distinctly showed by qPCRand a72kD fusion protein was also detected by Western blot technique. CCK-8testshowed the cell growth in pGC-FU-WWOX group of both Jurkat and K562cells wereinhibited remarkably than that of two control groups(P＜0.05). Clone forming assayrevealed the cloning formation ability of pGC-FU-WWOX group cells of both Jurkatand K562cells were significantly decreased. There were obvious ultrastructurechanges of pGC-FU-WWOX group cells in Jurkat and K562cells with the morpha ofchromatin condensation, shrink in phrase Ⅱ b exhibited by DAPI stain. DNAdegradative fragments in pGC-FU-WWOX group of Jurkat and K562cells weredetected by DNA ladder electrophoresis, which also showed apoptosis degree ofpGC-FU-WWOX group increased with time prolonged. Annexin V PE/7-AADfluorescence test via FCM showed cells in pGC-FU-WWOX groups of Jurkat andK562had a significant high apoptosis rate than that of two control groups (P＜0.001),which most of the cells were in late apoptosis stage.3. Indirect immunofluorescenceunfolded that cytochrome C of pGC-FU-WWOX group located in cytoplasm with adispersive or block like distribution both in Jurkat and K562cells, while p73, p53andNF-κB p65located in cytoplasm equably with no difference. qPCR and Western blotrevealed that compared with the other two control groups the expression of Bcl-2protein in pGC-FU-WWOX group of Jurkat and K562cells decreased in atime-dependent manner and cytochrome C increased as well as the activation ofCaspase-9and Caspase-3compared with two control groups, while p53changedunconspicuously. Bax protein of pGC-FU-WWOX group ascended only observed inK562cells. SDS-PAGE of pGC-FU-WWOX groups in Jurkat and K562cells exhibitsa high concentration protein mixture on73～74kD of the marker afterimmunoprecipitation, while the control groups were negative. At the same time,WWOX and p73proteins were positive in the immunoprecipitation mixture of the target gene groups with the help of Western blot, while p53protein was undetected.Conclusions:1. Lentivirus mediated-WWOX overexpression system wasconstructed successfully.2. Overexpression of WWOX gene exibits an antitumoreffect by inhibiting the proliferation of Jurkat and562cells and inducing theirapoptosis.3. Overexpression of WWOX gene in Jurkat and562cells coulddown-regulate the expression of Bcl-2and increase cytochrome C as well as activateboth Caspase-9and Caspase-3. By activating the endogenous mitochondria pathwaysis one of the possible mechanisms in its tumor suppression activities of WWOX inJurkat and K562cells.4. WWOX could combine with p73directly in its apoptosisinducing activities in Jurkat and K562leukemic cells.