乳腺癌MCF-7细胞系9p21缺失断点的精确定位分析

Deletion breakpoint mapping on chromosome 9p21 in breast cancer cell line MCF-7

目的对乳腺癌MCF-7细胞系9p21区缺失断点进行精确定位。方法采用多重连接探针扩增技术(MLPA)检测MCF-7细胞系9p21区缺失断点的大致区间,采用长PCR扩增缺失断点,引物步移缩小缺失断点的所在区间,测序确定缺失断点的位置。结果 MLPA检测探明MCF-7细胞系的端粒侧断点位于MTAP基因内,着丝粒侧断点位于CDKN2A基因内;通过长PCR、引物步移及测序确定在MCF-7细胞系中,缺失位点位于chr9:21819532至chr9:21989622之间,大小为170kb;断点端粒侧位于MTAP基因的内含子4内,着丝粒侧位于CDKN2A(p14)基因的内含子1内近外显子1β处。结论长PCR是缺失断点定位的良好方法。在MCF-7细胞系中,存在一个起于MTAP基因内、止于CDKN2A基因内、大小为170kb的缺失片段,其意义有待进一步研究。

Objective To map the deletion breakpoint of chromosome 9p21 in breast cancer cell line MCF-7.Methods The deletion of chromosome 9p21 was checked by Multiplex Ligation-dependent Probe Amplification（MLPA） in MCF-7. Subsequently,the deletion breakpoint was amplified by long range PCR and the deletion region was narrowed by primer walking. Finally,the deletion position was confirmed by sequencing.Results The deletion was found starting within the MTAP gene and ending within CDKN2A gene by MLPA.Based on long range PCR and primer walking,the deletion was confirmed to cover the region from chr9：21819532 to chr9：21989622 by sequencing,with a deletion size of 170kb,starting within the intron 4 of MTAP and ending within the intron 1 near exon 1β of CDKN2A.Conclusions Long range PCR is an efficient way to detect deletion breakpoints. In MCF-7, the deletion has been confirmed to be 170kb, starting within the MTAP gene and ending within the CDKN2A gene.The significance of the deletion warrants further research.