Mimori Koshi, Kataoka Akemi, Yoshinaga Keiji, Ohta Mitsuhiko, Sagara Yasuaki, Yoshikawa Yasuji, Ohno Shinji, Barnard Graham F, Mori Masaki
Department of Surgery Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan.
Clin Exp Metastasis. 2005;22(1):59-67. doi: 10.1007/s10585-005-4417-y.
Comparing differential gene expression profiles established by cDNA microarray between normal cells (N), primary carcinoma cells (T), and metastatic carcinoma cells (M) may determine those critical genes directly associated with progression and metastasis of breast cancer. Total RNA was extracted by laser microdissection (LMD) from 20 slices of T, N and M from 6 cases. After amplification by a T7-based system, differentially expressed genes between T, N and M were identified by cDNA microarray. In addition, to clarify the mechanism for altered gene expression, we determined the methylation status by sequencing after bisulfite treatment for intriguing genes. As a result, the expression of motility related protein-1 (MRP-1/CD9), peripheral myelin protein-22 (PMP-22), and caspase 3 (CASP-3) were down-regulated in M compared to T. We focused especially on MRP-1 and found that the expression status of MRP-1 was significantly inversely associated with stage of disease in 56 cases of breast cancer (P<0.05), and the relapse free survival in 5 years was significantly higher in MRP-1 positive cases than those negative cases (P<0.05). Conversely, overexpression, by 11-fold, of signal transduction and translation factors were observed in T compared to N. The cancer specific methylation was observed only in CASP-3 in a case. In conclusion, the establishment of the present assay allows us to detect genes directly associated with each cell population within tumor tissue and gives us clues to identify metastasis-related genes comprehensively in clinical breast cancer cases.
比较通过cDNA微阵列建立的正常细胞（N）、原发性癌细胞（T）和转移性癌细胞（M）之间的差异基因表达谱，可能会确定那些与乳腺癌进展和转移直接相关的关键基因。通过激光显微切割（LMD）从6例患者的20片T、N和M组织切片中提取总RNA。经基于T7的系统扩增后，通过cDNA微阵列鉴定T、N和M之间差异表达的基因。此外，为了阐明基因表达改变的机制，我们对感兴趣的基因进行亚硫酸氢盐处理后通过测序确定甲基化状态。结果，与T相比，转移性癌细胞（M）中运动相关蛋白-1（MRP-1/CD9）、外周髓鞘蛋白-22（PMP-22）和半胱天冬酶3（CASP-3）的表达下调。我们特别关注MRP-1，发现在56例乳腺癌中，MRP-1的表达状态与疾病分期显著负相关（P<0.05），MRP-1阳性病例的5年无复发生存率显著高于阴性病例（P<0.05）。相反，与N相比，在T中观察到信号转导和翻译因子过表达11倍。在1例病例中仅在CASP-3中观察到癌症特异性甲基化。总之，本检测方法的建立使我们能够检测与肿瘤组织内每个细胞群体直接相关的基因，并为我们在临床乳腺癌病例中全面鉴定转移相关基因提供线索。
