Objective: To investigate the expression and potential role of heterogeneous nuclear ribonucleo-protein A2B1 (HNRNPA2B1) in mouse cerebellar development and the significance of HNRNPA2B1 in human medulloblastoma. Methods: The data of HNRNPA2B1 RNA expression in mouse and human cerebella were obtained from databases. Western blot and immunohistochemical staining were performed to detect the protein level of HNRNPA2B1 in mouse cerebella at different ages. The expression level of HNRNPA2B1 in control human cerebellum and medulloblastoma was detected by immunohistochemical staining. m6A-IP-qPCR method was applied to confirm whether HNRNPA2B1 RNA in Daoy cells was modified with m6A.Western blot was used to detect the effect of MG132 treatment on the HNRNPA2B1 protein level in Daoy cells. Results: The level of HNRNPA2B1 protein in postnatal mouse cerebella was higher than that in adult mouse cerebella, with weak HNRNPA2B1 staining in external granular cells while strong staining in mature Purkinje cells and molecular layer. Compared with control normal human cerebella, the RNA expression level of HNRNPA2B1 increased in medulloblastoma, while immunohistochemical staining showed that the mean intensity of HNRNPA2B1 decreased in medulloblastoma. HNRNPA2B1 RNA in medulloblastoma and Daoy cells was modified by m6A. The HNRNPA2B1 protein level in Daoy cells increased upon MG132 treatment. Conclusions: HNRNPA2B1 is dynamically expressed during mouse cerebellar development. Compared with normal human cerebella, HNRNPA2B1 is significantly up-regulated at transcriptional level but obviously down-regulated at translational level in medulloblastoma. These results indicate that HNRNPA2B1 may be involved in cerebellar development process and medulloblastoma tumorigenesis. The m6A methylation in HNRNPA2B1 transcript and protein ubiquitin-proteasome pathway may account for the down-regulation of HNRNPA2B1 at protein level.
目的： 探讨异质性细胞核核糖蛋白（HNRNP）A2B1在小鼠小脑发育及髓母细胞瘤中的表达模式及意义。 方法： 从数据库查询小鼠小脑及人脑中基因的RNA水平。免疫印迹实验与免疫组织化学实验检测不同年龄C57BL/6野生型小鼠小脑中HNRNPA2B1的蛋白水平。通过免疫组织化学实验检测对照人小脑样本与人髓母细胞瘤中HNRNPA2B1的蛋白水平。m6A-IP-qPCR检测Daoy细胞中HNRNPA2B1 RNA m6A甲基化水平；利用人髓母细胞瘤Daoy细胞检测蛋白酶抑制剂MG132对HNRNPA2B1蛋白丰度的影响。 结果： 小鼠小脑发育期HNRNPA2B1的RNA及总体蛋白水平高于成年小鼠小脑；原位水平上HNRNPA2B1低表达于含有增殖细胞的外颗粒层，而在浦肯野细胞与分子层细胞中高表达。HNRNPA2B1在髓母细胞瘤中的RNA水平显著高于正常人小脑，但免疫组织化学染色阳性强度明显降低，且差异具有统计学意义（P<0.05）。人髓母细胞瘤及Daoy细胞中HNRNPA2B1 RNA上含有m6A修饰；10 μmol/L或20 μmol/L的MG132处理均可引起Daoy细胞中HNRNPA2B1蛋白水平升高。 结论： HNRNPA2B1在小鼠小脑发育过程中的表达呈动态变化；与对照组相比，人髓母细胞瘤中HNRNPA2B1的RNA水平显著上升，而蛋白水平明显降低，提示HNRNPA2B1可能与小脑发育及髓母细胞瘤进程相关。HNRNPA2B1 RNA m6A修饰及蛋白的泛素化修饰可能是导致其在髓母细胞瘤中RNA丰度升高而蛋白水平降低的原因。.
Keywords: Animal testing alternatives; Cerebellar cortex; Medulloblastoma; Mice, Inbred C57BL; Ribonucleoproteins.