食管癌淋巴结转移相关基因筛选的研究

Screening of lymphatic metastasis-associated genes in esophageal squamous cell carcinoma

目的:利用cDNA microarray比较食管癌淋巴结转移与非转移患者的基因表达谱,旨在筛选食管癌淋巴转移相关基因,加深对其转移机制的了解。方法:按一步法抽提35例原发性食管癌和相应正常食管黏膜上皮细胞的总RNA并纯化mRNA;应用T7RNA聚合酶进行RNA扩增成cRNA,将等量的食管癌细胞和对应正常细胞cRNA分别逆转录合成荧光分子掺入的cDNA一链做探针,混合后杂交于癌基因芯片。严格洗片后杂交信号经扫描等处理并用生物信息学对检测结果进行分析。同时应用实时荧光定量RT-PCR进行验证试验。结果:58条基因在食管癌淋巴结转移与非转移患者之间差异有统计学意义(P<0.05),其中上调表达基因28条,主要集中于细胞黏附分子和细胞受体,下调基因30条,主要为细胞周期调节和细胞间信号分子。实时荧光定量RT-PCR验证其中6个基因,结果一致。结论:高通量基因芯片技术筛选出大量食管癌淋巴结转移相关基因,对这些基因功能的验证有助于找到淋巴转移的关键基因或通路,为预防和控制其转移提供了新的思路与线索。

OBJECTIVE： To screen the differentially expressed genes between primary esophageal squamous cell carcinoma （ESCC） with and without lymphatic metastasis by cDNA microarray, and to understand the mechanism of lymphatic metastasis. METHODS： The total RNA was extracted from the cells obtained by means of LCM from the primary carcinoma, the corrresponding esophageal epithelium in 35 cases of ESCC, and purified to mRNA by Oligotes. RNA was amplified by the T7-based amplification system and then applied to a cDNA microarray. Both cRNAs from the esophageal and normal esophageal mucosa were reversely transcribed to the cDNA with the incorporation of fluorescent dUTP to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After the high-stringent washing, the cDNA microarray was scanned for the fluorescent signals, and the lymphatic metastasisassociated genes were screened by bioinformatics between ESCC samples with and without lymphatic metastasis. Six genes were selected and validated with real time quantitative RT-PCR （qRT-PCR）. RESULTS： Fiftyeight genes significantly differed between the ESCC with and without lymphatic metastasis （P〈0.05）, of which 28 were up-regulated and 30 were down-regulated. The up-regulated genes were involved in cell adhesion molecules and cell membrane receptors and the down-regulated genes were mostly cell cycle regulators and intracellular signaling molecules. The expressions of 6 genes validated by qRT-PCR were concordant with those detected by cDNA microarray. CONCLUSION： Many lymphatic metastasisassociated genes in ESCC are screened by high-throughput gene chip, and validating their cellular functions will help to identify the key gene or pathway responsible for lymphatic metastasis, which might open up a new idea or a clue for preventing and controling lymphatic metastasis in ESCC.