O-GlcNAcylation通过调节Rack1蛋白稳定性参与SHH型髓母细胞瘤的形成

O-GlcNAcylation participates in initiation of SHH-type medulloblastoma by regulating Rack1 protein stability

    摘要
  • 摘要:
    目的 研究O-GlcNAcylation调节蛋白激酶C受体1（receptor for activated C kinase 1，Rack1）的稳定性在SHH型髓母细胞瘤（SHH type medulloblastoma，SHH-MB）形成中的功能作用。
    方法 选取中国人民解放军西部战区总医院临床肿瘤标本库中分子分型所确定的SHH-MB肿瘤及癌旁组织，分析样本中Rack1和O-GlcNAcylation（O-GlcNAc）的表达水平差异。对于人源髓母细胞瘤细胞系Daoy使用糖基化转移酶（OGT）抑制剂（OSMI-1）和去糖基化转移酶（OGA）抑制剂（TM-G）进行处理，通过Cell Counting Kit-8（CCK-8）法和免疫荧光染色检测肿瘤细胞增殖能力。采用O-GlcNAc酶标记系统、免疫共沉淀（Co-IP）和Western blot法判断Rack1有无发生O-GlcNAc，而后通过环己酰亚胺（CHX）实验和泛素化修饰实验证实O-GlcNAcylation对Rack1蛋白水平的影响。构建敲低Rack1的髓母细胞瘤模型，通过Cell Counting Kit-8（CCK-8）法、免疫荧光染色和划痕实验检测肿瘤细胞增殖能力。同时通过在免疫缺陷型小鼠进行异种原位肿瘤移植进行验证，在所得组织样本中（sh-NC和sh-Rack1）使用Western blot检测下游SHH信号通路变化。
    结果 Rack1和O-GlcNAcylation在SHH-MB中表达水平显著增高，且Rack1表达水平和患者生存率呈负相关关系。对Daoy细胞系使用OSMI-1、TM-G处理后，发现O-GlcNAc能明显促进Daoy细胞增殖，而抑制细胞O-GlcNAc则抑制细胞增殖。分子实验证实Rack1蛋白O-GlcNAcylation可以调节其蛋白稳定性，进而促进肿瘤细胞增殖。在Daoy细胞系敲低Rack1表达，其细胞增殖能力明显低于对照组；在动物水平方面，相较于对照组，Rack1蛋白敲低的肿瘤组织增殖受到显著抑制。并且Rack1可通过调节SHH信号通路参与SHH-MB形成。
    结论 O-GlcNAcylation可通过调节Rack1蛋白的稳定性进而参与SHH-MB形成。

     

    Abstract:
    Objective To investigate the role of O-GlcNAcylation in the regulation of the stability of receptor for activated C kinase 1 (Rack1) during SHH-type medulloblastoma (SHH-MB) initiation.
    Methods SHH-MB tumors and adjacent tissues were selected from the clinical tumor specimen library of the Department of Pathology at The General Hospital of Western Theater Command. Rack1 expression and O-GlcNAcylation (O-GlcNAc) levels in these tumor tissues were analyzed. The human medulloblastoma cell line Daoy was treated with a glycosyltransferase (OGT) inhibitor (OSMI-1) and adeglycosyltransferase (OGA) inhibitor (TM-G), and their impact on tumor cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8) and immunofluorescence staining. The O-GlcNAc enzyme labeling system co-immunoprecipitation (Co-IP) and Western blot were used to correlate Rack1 protein levels with O-GlcNAc levels. The impact of O-GlcNAcon Rack1 stability was confirmed using cycloheximide (CHX) and ubiquitination modification experiments. In a medulloblastoma mouse model with Rack1 knockdown, tumor cell proliferation was detected using a Cell Counting Kit-8 (CCK-8), immunofluorescence staining, and a scratch assay. Xenograft tumors were transplanted into immunodeficient mice and SHH signaling was detected by Western blot in the obtained tissue samples (sh-NC and sh-Rack1) to verify the role of Rack1 protein in SHH-MB.
    Results Rack1 and O-GlcNAcylation levels were significantly increased in SHH-MB tumor samples, and a negative correlation was observed between Rack1 levels and patient survival rates. Treatment of Daoy cells with OGT and TM-G revealed that O-GlcNAc significantly promotes Daoy cell proliferation, while inhibiting O-GlcNAc impedes tumor cell proliferation. Molecular experiments have confirmed that O-GlcNAc modification of Rack1 protein can regulate tumor cell stability, thereby promoting tumor cell proliferation. When Rack1 expression was knocked down in Daoy cells, cell proliferation was significantly reduced relative to control cells. Accordingly, proliferation was significantly inhibited in tumor tissues with Rack1 protein knockdown in mouse models, suggesting that Rack1 can participate in the initiation of SHH-MB by regulating SHH signaling.
    Conclusions O-GlcNAcylation participates in SHH-MB initiation by regulating Rack1 stability.

     

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