磷酸化修饰增强Sufu对髓母细胞瘤的抑癌作用
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国家自然科学基金面上项目(81572720,81871936);国家自然科学基金青年科学基金项目(81702747,81602431)


Phosphorylation enhances the inhibitory effect of Sufu on medulloblastoma
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    摘要:

    目的:探讨在髓母细胞瘤(medulloblastoma,MB)中磷酸化修饰对Suppressor of fused(Sufu)抑癌活性的调节和潜在机制。方法:免疫组化分析MB组织芯片中Sufu的表达及磷酸化水平。在MB细胞系DAOY中转染野生型Sufu或其磷酸化位点突变质粒,分别通过细胞免疫化学染色、Western blot、CCK8、EdU标记、细胞流式检测等分析Sufu的核浆分布以及转染细胞的增殖、凋亡等指标,通过荧光素酶报告基因实验检测转染细胞Hedgehog(Hh)信号通路下游胶质瘤相关癌基因同源物(hlioma-associated oncogene homologue,Gli)的转录活性,Western blot检测鸟氨酸脱羧酶(ornithine decarboxylase,ODC1)的表达水平。结果:Sufu在MB组织的表达明显高于正常小脑,并呈现磷酸化的修饰状态。磷酸化Sufu主要聚集在细胞核。Sufu能抑制DAOY细胞的增殖及克隆形成,促进细胞凋亡。非磷酸化的Sufu突变体抑癌作用明显减弱。已知在Hh通路下游,Gli介导的转录活化以及ODC1介导的多胺合成增加均可以促进MB细胞的生长。磷酸化Sufu抑制Gli介导的转录活化,非磷酸化Sufu对Gli的抑制作用减弱,并促进ODC1的表达。结论:Sufu发生磷酸化修饰后,对MB的抑制作用增强,其机制可能与抑制Hh信号下游的Gli转导活性和细胞的多胺代谢有关。

    Abstract:

    Objective:This study aims to investigate how phosphorylation regulates the tumor-suppressive activity of Suppressor of fused(Sufu)in medulloblastoma(MB) cells and elucidate the potential mechanism. Methods:The expression and phosphorylation levels of Sufu in MB tissue microarrays were detected by immunohistochemistry. Wild type Sufu or its phosphorylation sites mutated plasmids were transfected into MB cell line DAOY. The cellular distribution of Sufu was detected by ICC and Western blot,and the cell proliferation and apoptosis were detected by CCK8,EdU labeling,and flow cytometry. Besides,the transcriptional activity of Glioma-associated oncogene homologue(Gli) at downstream of Hedgehog(Hh) signaling was analyzed by luciferase reporter assay,and the protein level of ornithine decarboxylase(ODC1)was detected by Western blot in transfected cells. Results:Sufu was highly expressed and phosphorylated in MB tissues,compared to normal cerebellum. Phosphorylated Sufu was mainly concentrated in the nucleus. Unphosphorylation dampened the inhibitory effect of Sufu on proliferation and its promotion of apoptosis in DAOY cells. It is known that Gli-mediated transcriptional activation and ODC1-mediated polyamine synthesis can promote MB cell growth at the downstream of Hh signaling. Phosphorylated Sufu inhibited the transcriptional activity of Gli. Unphosphorylated Sufu showed impaired repressor activity and promoted the expression of ODC1. Conclusion:Phosphorylation enhances the inhibitory effect of Sufu on MB cell growth,which may be related to inhibition of Gli-mediated Hh signaling transduction and the metabolism of polyamines.

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廖恒青,濮晓红,沈龙艳,蔡 静,刘 晨,俞婷婷,乐 珅,程 雁.磷酸化修饰增强Sufu对髓母细胞瘤的抑癌作用[J].南京医科大学学报(自然科学版),2019,(8):1151-1158

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  • 收稿日期:2019-02-11
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  • 在线发布日期: 2019-08-29
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