癌基因B—RafV600E导致黑色素瘤Sbc12和SK—MEL31细胞染色体不稳定性的分子机制研究

Molecular mechanism of oncogenic B-RafV600E induces chromosome instability in melanoma Sbcl2and SK-MEL31 cells

目的 研究癌基因B-RafV600E导致肿瘤细胞染色体不稳定性的分子机制。方法 采用RNAi技术敲除稳定表达B-RafV600E基因的Sbcl2和SK-MEL31细胞中内源性Mps1基因表达，免疫荧光染色技术显示中心体及纺锤体结构。HU-arrest assay观察Mps1基因缺失对癌基因BRAFV600E导致肿瘤细胞中心体过度复制及多极纺锤体形成的影响。结果 未敲除内源性Mps1基因的表达B-RafV600E基因的Sbcl2和SK-MEL31细胞中约有36%出现中心体过度复制及多级纺锤体，但当Mps1基因被敲除后上述异常细胞降低至6%。结论 B-RafV600E可能通过Mps1调控中心体过度复制及多极纺锤体结构的形成，进而影响肿瘤细胞染色体不稳定性及非整倍体细胞的出现。

Objective To explore the molecular mechanism of BRAFV600E inducing chromosome instability in melanoma Sbcl2 and SK-MEL31 cells. Methods Using siRNA approach to deplete the endogenous expression of Mps1 in stable Sbcl2 and SK-MEL 31 BRAFV600E expression clones and immunofluoresence to assess centorosmes and mitotic spindles. Using HU-treatment to arrest cells at S-phase and test the effect of BRAFV600E on centrosome amplification and the formation of multipolar spindles. Results The percentage of BRAFV600E expressing Sbcl2 and SK-MEL31 cells with centrosome amplification and multipolar spindle was reduced from 36% to 6% when Mps1 was absent. Conclusion The results indicate that BRAFV600E maybe regulates centrosome amplification and multipolar spindle through Mps1, thus leads to chromosome instability in tumor cells.