利用mRNA差异显示和基因芯片技术联合筛选乳腺原发癌与淋巴结转移癌患者的差异表达基因

目的 通过乳腺原发癌与配对的淋巴结转移癌基因表达谱的比较研究筛选乳腺癌转移相关基因。方法 首先利用mRNA差异显示（mRNA DD）技术筛选乳腺原发癌与其配对的淋巴结转移癌的差异表达基因片段,将差异基因片段克隆、测序并与GenBank同源性比较;然后利用同位素标记的反向斑点杂交和荧光标记的基因芯片杂交方法验证筛选得到的差异基因;采用实时定量逆转录聚合酶链反应（real-time RT—PCR）方法检测差异表达基因在7个不同生物学行为乳腺癌细胞系的mRNA表达。结果 乳腺癌的淋巴结转移癌与其原发癌的基因表达谱具有高度一致性,但也有少量差异表达的基因;mRNADD筛选得到16个差异基因,包括4个未知基因并已登录在GenBank,序列号为BG518428、BG518429、BM005520、BM005521,4个已知序列未知功能基因和8个已知基因。其中驱动蛋白样DNA结合蛋白（KNSL4）和二氢嘧啶脱氢酶（DYPD）为在同位素标记的反向斑点杂交和基因芯片杂交的验证结果中证实在转移癌中的表达较原发癌下调的基因。KNSL4 mRNA在7个不同生物学行为乳腺癌细胞系的表达,随着细胞转移能力的增强呈下调趋势。结论 乳腺原发癌与配对的淋巴结转移癌基因表达谱的相似性证实,淋巴结转移癌是其原发癌的转移亚克隆,二者的差异表达基因可能与细胞的转移表型相关;KNSL4和DYPD为3种方法均证实的在转移癌中表达下调的基因,是潜在的乳腺癌转移相关基因;KNSL4与乳腺癌细胞转移的生物学行为相关,提示其可能在乳腺癌转移中起重要作用。

Identification of the differentially expressed genes between primary breast cancer and paired lymph node metastasis through combining mRNA differential display and gene microarray

Objective To identify and clone the genes related to breast cancer metastasis through comparing the mRNA expression profiling between primary breast cancer and paired lymph node metastasis. Methods First,primary breast cancer cells and paired lymph node metastasis tissues were used to identify their differentially expressed genes by using mRNA differential display,and fragments of differentially expressed genes were obtained by gel cutting and cloning to pGEM-T vector,then sequencing and blasting with the GenBank for their homologous genes.Then,the obtained genes were validated by using reverse dot blot hybridization and gene microarray.Finally,the mRNA expression levels in the 7 breast cancer cell lines with different metastasis behaviors were detected by real-time RT-PCR.Results The mRNA expression profiling of the metastatic breast cancers in lymph node was similar to that of their primary cancers.16 differentially expressed gene fragments were obtained from mRNA differential display;four of them were found to be unknown genes,and had been accepted by the GenBank,with the accession numbers BG518428,BG518429,BM005520,and BM005521.Of the other 12 genes,8 were known genes,and 4 were genes with known sequences but unknown functions.The kinesin-like DNA binding protein(KNSL4) and dihydropyrimidine dehydrogenase(DYPD)were down-regulated in the metastatic tissues in comparion with the paired primary tissues,which was identified by both radioactivity-labeled reverse dot blot hybridization and fluorescence-labeled gene microarray hybridization.KNSL4 mRNA expression had a down- regulation trend with increasing metastatic ability of breast cancer cell lines.Conclusions The gene expression profiling of the lymph node metastasis is almost similar to that of their primary breast cancer, indicating that the metastatic cancer cells in lymph node are the subclone with higher metastasis ability of the primary cancer cells,and that some differentially expressed genes between them may be involved in the change of metastasis phenotype.KNSL4 and DYPD are potential metastasis-related genes,and KNSL4 mRNA expression is correlated with metastasis behaviors of the breast cancer cell lines,suggesting that KNSL4 may play an important role in the metastasis process of breast cancer.