Rock2调控O-GlcNAc糖基化在骨肉瘤生物靶向治疗药物TRAIL耐药性的机制研究

TRAIL resistance is the main problem in the treatment of osteosarcoma(OS),but its mechanism is unclear. It has been reported that O-GlcNAc glycosylation ​played an important role in TRAIL resistance. Our previous study finds that both ROCK2 and O-GlcNAcylation are highly expressed in OS tissues. Moreover, in OS cells, decrease of ROCK2 can significantly inhibit cell proliferation and enhance TRAIL-induced cell apoptosis. Meanwhile O-GlcNAcylation is also significantly reduced. we further show that O-GlcNAc transferase (​OGT) is decreased when reducing ROCK2, but this pathway will be blocked after decreasing proteasomes degradation function. Therefore, we hypothesized that Rock2 promotes over-expression of O-GlcNAcylation by inhibiting OGT ubiquitination/degradation, which eventually leads to TRAIL resistance of OS. Our study firstly uses the vitro/vivo experiments to clarify the role of ROCK2 in TRAIL resistance of OS, and reveals a mechanism that ROCK2 induces TRAIL resistance by regulating O-GlcNAcylation. Finally, we make the correlation analysis between ROCK2/ O-GlcNAcylation levels and the clinicopathologic characteris in OS tissues. However, our study on molecular biology mechanism of OS may provide a more effective therapeutic target.

TRAIL耐药是导致骨肉瘤（OS）靶向治疗失败的主要原因，但具体机制不清。研究报道O-GlcNAc糖基化在肿瘤TRAIL耐药中发挥重要作用。前期研究发现：ROCK2和O-GlcNAc糖基化在OS组织中均呈高表达；下调Rock2可以抑制OS细胞增殖，增强TRAIL诱导的OS 凋亡，O-GlcNAc糖基化也明显降低；进一步发现，降低 ROCK2的表达，糖基化转移酶OGT下降，抑制蛋白酶体降解可以阻断ROCk2对OGT的调控作用。据此，我们推测Rock2通过抑制OGT的泛素化降解促进O-GlcNAc糖基化从而导致OS对TRAIL耐药。本项目拟先利用体内外实验明确ROCK2在OS细胞TRAIL耐药中的作用，阐明ROCK2调控细胞O-GlcNAc糖基化导致对TRAIL耐药的机制，最后分析骨肉瘤组织中ROCK2及O-GlcNAc糖基化水平与患者临床病理特征的关联。本研究将为OS临床治疗提供一种新途径。

骨肉瘤是一种常见的骨肿瘤，预后较差。因此，需要新的组合疗法，使抗癌药物抗性骨肉瘤细胞对肿瘤坏死因子相关的凋亡诱导配体 (TRAIL) 敏感。 GTPase RhoA 效应器，Rho 相关的卷曲螺旋形成蛋白激酶 2 (ROCK2)，因其在各种癌症中的作用而广为人知。然而，尚未仔细研究其参与骨肉瘤。在这项研究中，我们分析了骨肉瘤患者的 ROCK2 表达、临床病理学特征和预后。分别使用流式细胞术、集落形成测定和 CCK8 测定分析细胞凋亡、集落形成和细胞增殖。蛋白质组学分析用于评估骨肉瘤的进展。我们发现邻近组织的 ROCK2 表达水平低于骨肉瘤组织，并且表达水平与骨肉瘤肿瘤大小和预后有关。骨肉瘤预后与 ROCK2 表达水平相关，ROCK2 表达水平可作为多变量分析的独立标志物。 ROCK2 沉默抑制体内和体外增殖并引发凋亡性骨肉瘤细胞死亡。 ROCK2 抑制骨肉瘤细胞中 TRAIL 介导的凋亡途径并促进活化。从机制上讲，ROCK2 通过 O-GlcNAc 转移酶降解改变 O-GlcNAc 化来影响骨肉瘤进展和 TRAIL 抗性。总之，我们的结果证明了一种独特的机制，即 ROCK2 影响骨肉瘤进展和 TRAIL 抗性，从而改善骨肉瘤预后。