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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,将差异基因片段克降、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;mRNA DD 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。
OBJECTIVE: To screen breast cancer metastasis-associated genes by comparative study of primary breast cancer and paired lymph node metastasis oncogene expression profiles. Methods Firstly, mRNA differential display (mRNA DD) was used to screen differentially expressed genes of primary breast cancer and its matched lymph node metastatic cancer. The differential gene fragments were cloned and sequenced and compared with GenBank. The differentially expressed genes were identified by hybridization with dot blot and fluorescently labeled cDNA microarray. Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) was used to detect the expression of differentially expressed genes in seven different biological behaviors of breast cancer cells Department of mRNA expression. Results The lymph node metastasis of breast cancer was highly consistent with the gene expression profile of its primary cancer, but there were also a few differentially expressed genes. Sixteen differential genes, including four unknown genes, were screened by mRNA DD and registered in GenBank. BG518428, BG518429, BM005520, BM005521, 4 known unknown genes and 8 known genes. Among them, kinesin-like DNA binding protein (KNSL4) and dihydropyrimidine dehydrogenase (DYPD) were shown to be downregulated in metastatic carcinoma compared with primary cancers in the validation results of isotope-labeled reverse dot blot hybridization and microarray hybridization gene. The expression of KNSL4 mRNA in breast cancer cell lines of seven different biological behaviors showed a downward trend with the enhancement of cell metastasis. Conclusions The similarity between the primary breast cancer and paired lymph node metastasis oncogene expression confirmed that lymph node metastasis is the metastatic subclone of its primary cancer, and the differentially expressed genes may be related to the metastatic phenotype of the cell. KNSL4 and DYPD The three genes that are down-regulated in metastatic cancer are potential metastasis-related genes of breast cancer. KNSL4 is correlated with the biological behavior of breast cancer metastasis, suggesting that KNSL4 may play an important role in the metastasis of breast cancer.