Effection On Biological Characteristics Of U251 And U251 Derived Tumor Stem Cells After Inhibition Of Pygo2 Expression

Objective: To explore the role of Pygo2 in glioma U251 and U251 derived tumor stem cells and its possible mechanism.Methods: Knocking down of the expression of Pygo2 in U251 cells using lentivirally expressed si RNA. Infection efficiency of glioma cells were observed by fluorescence microscope. The relative expression of Pygo2、CD133、Nestin、Sox2、C-myc genes were quantified by RT-PCR, the expression of Pygo2、H3K4me2、H3K4me3 were detected by Western Blot. Cell proliferation assay was measured by the Cell Titer 96 Aqueous One solution reagent, and apoptosis was analyzed by flow cytometry. Glioma cancer stem-like cell, particularly the so-called cancer(tumor) stem cell or cancer-initiating cell, can be enriched from established cell line using tumor sphere culture(suspension culture).Result: Infection efficiency of glioma cells can reach more than 80%. RT-PCR revealed that the m RNA expression of Pygo2 was significantly silenced in Pygo2 interfering sequence(Pygo2 si RNA) group compared to negative control scrambled sequence(Control si RNA) group(P<0.05). Pygo2 si RNA inhibited the Pygo2 expression, whereas the Control si RNA exhibited no effect. MTS proliferation assays showed that knockdown of Pygo2 expression led to significant proliferative suppression when compared with the control. After the cells were stained with Annexin V/7-ADD, significant increased of Pygo2 si RNA cells(early and late apoptosis) was observed by flow cytometry analysis compared with the control(P<0.05). Western blot showed the decreased H3K4me3 expression but not H3K4me2 in Pygo2 si RNA group cell. Silencing Pygo2 reduced Sox-2, Nestin, and CD133 m RNA levels in U251, which reportedly mark Neural Stem Cells(NSCs),as well as Wnt signaling downstream target genes C-myc. We detected higher levels of Pygo2 m RNAs in tumor spheres of U251 than in corresponding adherent cultures using quantitative RT-PCR. Knocking down Pygo2 expression resulted in a reduced number as well as size of tumor spheres.Conclusions: 1、Lentivirus vector mediated transfered into cells has high infection and interference efficiency.2、Pygo2 si RNA inhibite the growth and increase apoptosis of U251 cell by suppressing histone H3K4 trimethylation.3、Pygo2 may play an important role in self-renew and proliferation in U251 glioma cancer stem-like cells.