YBX1截短体抑制髓母细胞瘤DAOY的迁移和干性增殖能力  

The truncated YBX1 inhibits the migration and stem cell proliferation of medulloblastoma DAOY

作　　者：赖俊伟 叶子 俞婷婷[1] 程雁[1] LAI Junwei;YE Zi;YU Tingting;CHENG Yan(Department of Medical Genetics,School of Basic Medical Science,Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京医科大学医学遗传学系,江苏南京211166

出　　处：《生物技术》2024年第3期304-310,352,共8页Biotechnology

基　　金：国家自然科学基金面上项目(81261120386)。

摘　　要：[目的]利用CRISPR/Cas9技术构建YBX1基因截短的SHH型髓母细胞瘤DAOY细胞系,探讨截短的YBX1对DAOY细胞迁移和干性增殖的影响。[方法]设计针对YBX1基因的sgRNA序列,并将其插入pX330载体构建sgYBX1表达质粒。DAOY细胞经pX330-sgYBX1和耐青霉素质粒转染,药筛后采用极限稀释法挑取单克隆细胞系,使用测序及蛋白质印迹技术检测YBX1基因编辑的情况,利用免疫荧光实验检测YBX1截短体在DAOY细胞内的定位情况,并通过CCK8、Transwell、克隆形成实验、低黏附板成球实验检测YBX1截短体对DAOY细胞增殖、迁移及干性的影响。[结果]测序结果表明,在DAOYYBX1-trunc细胞系中,YBX1基因编码区缺失144个碱基,导致合成肽段的第21~68氨基酸(位于A/P domain和CSD结构域)缺失。免疫荧光实验结果表明截短的YBX1蛋白定位富集在细胞核内。与未编辑细胞相比,DAOYYBX1-trunc细胞的增殖能力基本不变,但迁移能力减弱了50%,克隆形成能力削减了54%,细胞成球能力削减了68%。[结论]成功构建YBX1蛋白在21~68氨基酸截短的DAOY细胞系,其削弱了DAOY细胞的迁移及干性增殖能力。[Objective]To construct a SHH medulloblastoma DAOY cell line with truncated YBXI gene using CRISPR/Cas9 technology,and to investigate the effect of truncated YBX1 on the migration and stem-like proliferation in DAOY cells.[Method] The sgRNA sequence for YBXI gene was designed and inserted into pX330 vector to construct sgYBX1-expressing plasmids.DAOY cells were transfected with pX330-sgYBXI,together with the puromycin-resistant plasmid.Monoclonal cell lines were selected by limiting dilution after puromycin screening,sequencing and Western Bltting technology were used to detect the editing of YBX1 gene.The localization of truncate YBX1 in DAOY cells was detected by immunofluorescence assay,and the effects of YBX1 truncation on the proliferation,migration and stemness of DAOY cells were detected by CCK8,Transwell,clone formation assay and low adhesion plate spheronulation say.[Result]Sequencing results indicate that in the DAOY YBXI-mune cell line,144 base pairs were deleted in the coding region of the YBX1 gene,it leads to the deletion of amino acids 21 to 68 in the synthesized peptide segment(located in the A/P domain and CSD structural domain).Immunofluorescence experiments reveal that the truncated YBX1 protein was predominantly localized within the cell nucleus.Compared to unedited cells,DAOYBXi-une cells showed no significant change in proliferative capacity but exhibited a 50%reduction in migration ability,a 54%decrease in clonogenic ability,and a 68%reduction in sphere-forming capacity.[Conclusion]A DAOY cell line with a truncated YBX1 protein,successfully constructed at amino acids 21-68,can weaken the migration and stem-like proliferative ability of DAOY cells.

关 键 词：YBX1基因 DAOY SHH型髓母细胞瘤 冷休克结构域 截短体 细胞定位 迁移 干性增殖 

分 类 号：Q23[生物学—细胞生物学]