背景:Rab相互作用溶酶体蛋白(RILP)含有α-螺旋线圈,在骨肉瘤中具有未探索的生物学功能。本研究研究了RILP在骨肉瘤细胞和组织中的表达,以确定RILP对骨肉瘤细胞生物学行为的影响及其潜在机制。
方法:肿瘤免疫评估资源(TIMER)数据库,将癌症基因组图谱(TCGA)数据库和基因表达综合(GEO)数据库用于生物信息学分析。免疫共沉淀实验用于确定两种蛋白质是否相互作用。在功能测试中,细胞计数试剂盒-8(CCK-8)测定,集落形成试验,伤口愈合试验,transwell侵袭试验,进行免疫荧光(IF)测定和免疫组织化学(IHC)测定。
结果:过表达RILP显著抑制骨肉瘤细胞增殖和转移能力受损,而RILP的沉默表现出相反的趋势。在143B细胞中应用RNA-seq数据分析,途径富集分析显示差异表达基因主要富集在PI3K/AKT途径中。我们进一步证实RILP过表达抑制了PI3K/AKT/mTOR信号通路并诱导骨肉瘤细胞自噬,而当使用PI3K通路激活剂740Y-P时,观察到相反的趋势。3-甲基腺嘌呤(3-MA),选择性自噬抑制剂,部分减弱了RILP对骨肉瘤细胞迁移和侵袭能力的抑制作用,提示自噬参与骨肉瘤细胞的上皮-间质转化调控。生长因子受体结合蛋白-10(Grb10),一种衔接蛋白,被证实为RILP抑制PI3K/AKT信号通路的潜在靶点。我们将稳定过表达143B骨肉瘤细胞皮下注射到裸鼠中,并观察到RILP的过表达通过抑制PI3K/AKT/mTOR途径来抑制肿瘤生长。
结论:我们的研究表明,RILP的表达与骨肉瘤的良好预后有关,RILP抑制增殖,迁移,通过Grb10介导的PI3K/AKT/mTOR信号通路的抑制,促进骨肉瘤细胞的侵袭和自噬。在未来,靶向RILP可能是骨肉瘤治疗的潜在策略.
Rab-interacting lysosomal protein (
RILP) contains an alpha-helical coil with an unexplored biological function in osteosarcoma. This study investigated the expression of
RILP in osteosarcoma cells and tissues to determine the effect of
RILP on the biological behaviors of osteosarcoma cells and the underlying mechanism.
Tumor Immune Estimation Resource (TIMER) database, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database were used for bioinformatic analysis. Co-immunoprecipitation experiment was used to determine whether the two proteins were interacting. In functional tests, cell counting kit-8 (CCK-8) assay, colony formation assay, wound healing assay, transwell invasion assay, Immunofluorescence (IF) assay and immunohistochemical (IHC) assay were performed.
Overexpression of RILP significantly inhibited proliferation and impaired metastasis ability of osteosarcoma cells, while silencing of
RILP showed the opposite trend. RNA-seq data analysis was applied in 143B cells and pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in the PI3K/AKT pathway. We further verified that overexpression of RILP restrained the PI3K/AKT/mTOR signaling pathway and induced autophagy in osteosarcoma cells, while the opposite trend was observed when PI3K pathway activator 740Y-P was used. 3-Methyladenine (3-MA), a selective autophagy inhibitor, partially attenuated the inhibitory effect of RILP on the migration and invasion ability of osteosarcoma cells, suggesting the involvement of autophagy in epithelial-mesenchymal transition regulation in osteosarcoma cells. Growth factor receptor binding protein-10 (Grb10), an adaptor protein, was confirmed as a potential target of RILP to restrain the PI3K/AKT signaling pathway. We subcutaneously injected stably overexpressing 143B osteosarcoma cells into nude mice and observed that overexpression of RILP inhibited tumor growth by inhibiting the PI3K/AKT/mTOR pathway.
Our study revealed that the expression of RILP was associated with favorable prognosis of osteosarcoma and RILP inhibits proliferation, migration, and invasion and promotes autophagy in osteosarcoma cells via Grb10-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. In the future, targeting
RILP may be a potential strategy for osteosarcoma treatment.