Glioblastoma (GB) is the most undifferentiated and aggressive central nervous system tumor , and its pathogenic mechanisms remain poorly understood. In our previous study, YWHAG gene overexpression was postulated as biomarker of bad prognosis using human GB tissue samples. YWHAG encodes for the γ member of the 14-3-3 family of intracellular dimeric phosphoserine-binding proteins that regulate signal transduction, cell cycle, metabolic cascades and antagonize apoptotic cell death in response to a triggering stimuli. This work aims to elucidate the mechanisms through YWHAG impacts in GB prognosis, to assess its potentiality as a therapeutic target.

YWHAG gene expression was silenced by siRNA in two IDH-wt GB cell lines, U251MG and LN229. YWHAG-silencing level was determined by western blot (WB) and RTqPCR. Cell proliferation and two-dimensional cell migration was measured by trypan blue exclusion and wound-healing assays respectively. Based on these findings, YWHAG expression was further silenced by a doxycycline-induced shRNA with an RFP reporter in U251MG. Transfection efficiency was evaluated by fluorescence using a LEICA DMI6000B microscope and LAS X software. YWHAG-silencing level was determined by RTqPCR. All experiments were performed at different time-points. Results were obtained by comparing non-target cells versus siRNA or shRNA -transfected cells in at least three independent experiments. Data was analyzed using GraphPad Prism 8 software.

In U251MG cells, the highest siRNA-YWHAG silencing rates were obtained at 72 h by RTqPCR (93.8%) and 96 h by WB (63.7%), and at 96 h by RTqPCR (84.3%) and WB (37%) in LN229. A statistically significant reduction of proliferation rate of U251MG siRNA-YWHAG was observed at 96 h after transfection (p-value=0.0079). A trend to a reduction in cell migration was also observed at 96 h after transfection. Regarding LN229, despite the reduction of cell migration 120 h after transfection (p-value=0.0001), no effect in proliferation was observed. Finally, U251MG cell line was selected for the establishment of the stable and inducible sh-YWHAG-silencing model. Suitable transfection rate by fluorescence signal was observed at 48 h after doxycycline treatment, with a promising 78% of gene silencing at RNA level in the sh-YWHAG cells.

YWHAG gene silencing causes a reduction in cell proliferation and migration in GB in-vitro models. To be deeply explored, a stable YWHAG inducible knocking-down model has been generated to confirm these results in preclinical advanced models, hence elucidating its potential role as biomarker and putative therapeutic target in GB.