采用单引物扩增法标记的基因芯片技术筛选乳腺原发癌与淋巴结转移癌的差异表达基因

目的比较乳腺原发癌与淋巴结转移癌的基因表达差异，筛选乳腺癌转移相关基因。方法采用单引物扩增(SPA)标记方法和含21000个人功能基因的Oligo芯片，比较10例淋巴结转移阳性乳腺癌患者的原发癌及淋巴结转移癌的基因表达差异，筛选至少5对样本中有1．5倍以上相同差异表达趋势的基因；采用实时定量RT—PCR方法对差异表达基因进行病例验证。结果SPA方法标记靶标可使用于一张芯片杂交的起始总RNA模板量减少至0．25μg；共筛选出57个基因在至少5对样本中有1．5倍以上相同的差异表达趋势，其中19个基因在转移癌中表达上调，38个基因下调，8个差异表达基因与细胞黏附和运动能力有关，14个与信号传导有关，14个与细胞生长代谢有关；实时定量RT—PCR检测30例乳腺癌原发癌与配对的淋巴结转移癌中纤维粘连蛋白(FN)的mRNA表达，转移癌中FN的mRNA表达平均相对表达量较原发癌下调3．6倍，配对t检验差异有统计学意义(t=-3．188，P=0．003)。结论(1)单引物扩增标记靶标的方法灵敏度高、重复性好。(2)以乳腺癌的淋巴结转移癌作为原发癌的转移亚克隆进行基因表达的差异比较，筛选得到的基因涉及了细胞黏附和运动能力、细胞信号传导、细胞生长代谢等与转移相关的生物学过程。(3)FN可能参与乳腺癌的转移过程，是潜在的预测乳腺癌转移和预后的分子标志。

Screening of differential expression genes between primary breast cancer and its lymph node metastasis using single primer amplification of cDNA for microarray

OBJECTIVE: To screen genes related to breast cancer metastasis by comparing difference of expression profile between primary breast cancer and its lymph node metastasis. METHODS: The cell total RNA was extracted from 10 breast cancer specimens, including the primary cancer and metastatic cancer tissues of axillary lymph nodes resected during operation. Single primer amplification (SPA) was used to prepare fluorescence-labeled targets and then Oligo microarray concluding 21 000 human functional genes was used to screen out 1.5 fold or more differential expression genes in at least 5 pairs of samples. The screened-out genes were identified by real-time PCR. RESULTS: Preparing targets by using SPA reduced the initial RNA to 0.25 microg, with 57 genes screened out, the expression of 19 of which were up-regulated and the expression of 38 of which were down-regulated in the metastatic tissues. Eight differentially expressed genes were related to cell migration and adhesion, 14 to signal transduction, and 14 to cell growth or metabolism. Real-time PCR showed that the fibronectin (FN) mRNA expression in the lymph node metastasis was only 1/3.6 of that in the matched primary breast cancer in 30 cases (t = -3.188, P = 0.003). CONCLUSION: SPA is a sensitive method for preparing targets. Genes related to cell migration and adhesion, signal transduction, and cell growth or metabolism, which are associated with the biology process of metastasis have been screened out. FN as a potent marker may contribute to diagnosis of metastasis and prognosis for breast cancer patients.