Background
Osteosarcoma is prevalent in children and adolescents. H1.4 modification is involved in various types of cancers. Ras pathway is often activated in human cancers. Herein, we explored the effects of Ras pathway through H1.4S35ph.
Conclusion
RasG12V/Y40C-PI3K/AKT signal pathway decreased H1.4S35ph through down-regulation of PKA while up-regulation of MDM2 in MG-63 cells. Highlights H1.4S35ph is regulated by K-RasG12V/Y40-PI3K/AKT in MG-63 cells; Overexpression of H1.4S35ph regulates MG-63 cell growth; H1.4S35ph regulates Ras downstream factors; K-RasG12V/Y40C-PI3K/AKT activity induces PKA degradation to down-regulate H1.4S35ph; K-RasG12V/Y40C-PI3K/AKT activity involves in PKA degradation via MDM2.
Methods
Osteosarcoma cancer cell line MG-63 was transfected with Ras gene with G12V and Y40C site mutation. The phosphorylation of H1.4S35 and AKT was detected by Western blot. Cell viability, cell colonies and migration were analyzed by MTT assay, soft-agar colony formation assay and Transwell assay, respectively. The expression of Ras pathway downstream factors and PKA was detected by qRT-PCR. The relationship between Ras and downstream factors was detected by ChIP. The cell cycle progression was measured by flow cytometry.
Results
Transfection with RasG12V/Y40C decreased H1.4S35ph expression while switched on p-AKTSer473. RasG12V/Y40C increased cell viability, colony numbers and migration while H1.4S35E (H1.4S35ph overexpression) led to the opposite results. The regulation of RasG12V/Y40C and H1.4S35E on Ras downstream factors was contrary to each other. Results demonstrated a positive relationship between PKA with H1.4S35ph with RasG12V/Y40C down-regulated both. However, PKA and MDM2 revealed negative regulation with RasG12V/Y40C transfection up-regulated MDM2.