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. 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. 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.

中文翻译：

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K-RasG12V / Y40C-PI3K / AKT途径通过PKA调节H1.4S35ph，促进骨肉瘤的发生和发展。

背景：骨肉瘤多见于儿童和青少年。H1.4修饰涉及各种类型的癌症。Ras途径通常在人类癌症中被激活。在这里，我们探讨了通过H1.4S35ph的Ras途径的影响。方法：用具有G12V和Y40C位点突变的Ras基因转染骨肉瘤癌细胞系MG-63。通过Western印迹检测H1.4S35和AKT的磷酸化。通过MTT测定，软琼脂菌落形成测定和Transwell测定分别分析细胞活力，细胞集落和迁移。用qRT-PCR检测Ras通路下游因子和PKA的表达。ChIP检测到Ras与下游因素之间的关系。通过流式细胞术测量细胞周期进程。结果：用RasG12V / Y40C转染可降低H1。开启p-AKTSer473时显示4S35ph表达。RasG12V / Y40C增加细胞活力，集落数和迁移，而H1.4S35E（H1.4S35ph过表达）导致相反的结果。RasG12V / Y40C和H1.4S35E对Ras下游因子的调控相互矛盾。结果表明，PKA与H1.4S35ph和RasG12V / Y40C均下调两者。然而，PKA和MDM2揭示了RasG12V / Y40C转染上调的MDM2的负调控。结论：RasG12V / Y40C-PI3K / AKT信号通路通过下调PKA而上调M-DM2在MG-63细胞中的表达而降低H1.4S35ph。亮点H1.4S35ph在MG-63细胞中受K-RasG12V / Y40-PI3K / AKT调控；H1.4S35ph的过表达调节MG-63细胞的生长。H1.4S35ph调节Ras下游因子；K-RasG12V / Y40C-PI3K / AKT活性诱导PKA降解，从而下调H1.4S35ph。K-RasG12V / Y40C-PI3K / AKT活性涉及通过MDM2降解PKA。