The truncated YBX1 inhibits the migration and stem cell proliferation of medulloblastoma DAOY
[Objective]To construct a SHH medulloblastoma DAOY cell line with truncated YBX1 gene using CRISPR/Cas9 technology,and to investigate the effect of truncated YBX1 on the migration and stem-like proliferation in DAOY cells.[Meth-od]The sgRNA sequence for YBX1 gene was designed and inserted into pX330 vector to construct sgYBX1-expressing plas-mids.DAOY cells were transfected with pX330-sgYBX1,together with the puromycin-resistant plasmid.Monoclonal cell lines were selected by limiting dilution after puromycin screening,sequencing and Western Blotting 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 arid stemness of DAOY cells were detected by CCK8,Transwell,clone formation assay and low adhesion plate spheronulation assay.[Result]Sequencing results indicate that in the DAOY YBX1-trunc 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,DAOYYBX1-trunc 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 prolifera-tive ability of DAOY cells.
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