核糖核苷酸还原酶M2在小脑髓母细胞瘤中的功能及机制

The biological function and mechanism of ribonucleotide reductase subunit M2 in medulloblastoma

目的:探讨核糖核苷酸还原酶M2(RRM2)在小脑髓母细胞瘤中的功能和机制。方法:分析GEO数据库中髓母细胞瘤患者的生存和RRM2表达水平的关系。通过免疫组化并结合数据库分析RRM2在小脑髓母细胞瘤和正常小脑组织中的表达。分别通过CCK-8细胞增殖实验、Transwell实验、流式细胞术检测RRM2对小脑髓母细胞瘤细胞系Daoy和D283的增殖、迁移、细胞周期和凋亡的调控作用。通过转录组测序(RNA-seq)探索下游分子机制。采用配对t检验和独立样本t检验进行统计学分析。结果:通过GEO数据库分析发现高表达RRM2的髓母细胞瘤患者相较于低表达组的总生存期更短、预后更差(P<0.05)。免疫组化及数据库分析结果显示RRM2在小脑髓母细胞瘤中的表达明显高于正常小脑组织(P<0.05)。与对照组比较,CCK-8细胞增殖实验、Transwell实验提示过表达RRM2促进肿瘤细胞增殖和迁移(P<0.05),敲低RRM2减少肿瘤细胞增殖和迁移(P<0.05);过表达RRM2能够明显增加S期的细胞比例(P<0.05),且明显减少细胞凋亡(P<0.05)。RNA-seq结果显示敲低RRM2后有3 454个基因表达上调,3 332个基因表达下调。KEGG分析显示RRM2敲低后多个信号通路受到影响,包括FOXO、细胞周期、核糖体生物合成等。结论:RRM2在小脑髓母细胞瘤中高表达且与预后不良有关,其具有促进小脑髓母细胞瘤细胞增殖、迁移,减少细胞凋亡的能力。

OBJECTIVE:To investigate biological functions and mechanism of ribonucleotide reductase subunit M2(RRM2)in medulloblastoma.METHODS:Relationships between survival and the levels of RRM2 expression in GEO database(GSE85217)were analyzed.Expressions of RRM2 in medulloblastoma and normal cerebellar tissues were analyzed using immunohistochemistry and database.CCK-8 proliferation experiments,transwell experiments,and flow cytometry were used to study the effects of RRM2 on proliferation,migration,cell cycle,and apoptosis of medulloblastoma cell lines Daoy and D283.RNA sequencing(RNA-seq)was used to explore downstream molecular mechanisms.Paired t-test and independent sample t-test were used for statistical analysis.RESULTS:Through the analysis of GEO database,survival of medulloblastoma patients was worse in the RRM2-high group than that in low group(P<0.05).The results of immunohistochemistry and database showed that expressions of RRM2 were significantly higher in medulloblastoma than that in normal cerebellar tissues(P<0.05).Compared to the control groups,overexpressions of RRM2 promoted tumor cell proliferation and migration,while knockdown of RRM2 reduced tumor cell proliferation and migration.In addition,overexpressions of RRM2 significantly increased the proportions of cells in the S phase(P<0.05)and reduced apoptosis(P<0.05).After knockdown of RRM2,RNA-seq results showed that 3454 genes were up-regulated and 3332 genes were down-regulated.KEGG analysis showed that multiple signal pathways were affected,including FOXO,cell cycle,ribosome biosynthesis,etc.CONCLUSION:RRM2 was highly expressed in medulloblastoma and the expression was negatively related to prognosis.In addition,RRM2 expression promoted proliferation and migration of medulloblastoma cells and reduced apoptosis.