The Effects Of Silencing USP22by SiRNA On Biological Behavior Of Primary Cultured GBM Cells

Background:Glioblastoma multiforme (GBM) is an aggressive primary brain tumour. A major challenge in patients with GBM is the nature of the tumor to invade into adjacent normal brain tissue which largely attributes to the poor prognosis of this disease. Invasive tumor cells can escape from surgical operation and are relatively resistant to radiation therapy and chemotherapy.The ubiquitin specific peptidase22(USP22) is an ubiquitin hydrolase, a key subunit of the human Spt-Ada-Gcn5acetyltransferase (SAGA) transcriptional cofactor complex, and has been recently identified as a member of11-gene Polycomb/cancer stem cell signature. Increasing experimental evidences indicate that USP22act as key factors in pathological processes of malignant epithelial and other solid tumors. It has already been reported that inhibition of USP22expression triggers cell cycle arrest in the human non-small cell lung carcinoma line H1299. However, the molecular mechanism is not clear yet. To our knowledge, the relationship between GBM and USP22remains unclear. A fully understanding of the role of USP22in the nature of GBM is critical for therapeutic strategies in the future. Objects and Methods:Part1:To identify whether USP22is involved in glioblastoma multiforme. The expression of USP22was assessed by RT-PCR and western blot analysis.Part2:To study the roles of USP22in primary cultured glioblastoma multiforme cells by silence USP22with symmetric interfering RNA.With the silence of USP22, the cell viability at different time points was examined by MTT assay. Cell cycle and cell apoptosis were evaluated by flow cytometric analysis.Results:1. Glioblastoma multiforme tissues express high levels of USP22.2. Primary cultured glioblastoma multiforme cells can be isolated successfully from fresh GBM tissues and cells are positive in immunofluorescence analysis of GFAP antibody.3. Three siRNA were synthesized targeting USP22and the most effective one was selected after comparation. USP22-specific siRNA down-regulates the expression of USP22successfully. MTT assays showed transfection with the USP22-specific siRNA inhibits the proliferation of GBM cells in vitro, compared with control siRNA.4. After transfection with USP22-specific siRNA, cell apoptosis was not triggered in our experiment, which indicated that a much more complex signal network might exist in the system.5. Silence of USP22expression induced GBM cell cycling arrest at G0/G1phases.6. Knowdown of USP22by the siRNA did not result in any change of p53, p21and cyclin E at mRNA level. Conclusions:USP22is up-regulated in glioblastoma multiforme tissues. Enzymatic digestion method can isolate primary cultured glioblastoma multiforme cells successfully. Transfection with the USP22-specific siRNA inhibits the proliferation of GBM cells in vitro through cell cycling arrest at G0/G1phases, though cell apoptosis was not triggered in our experiment.