Lechner S, Müller-Ladner U, Renke B, Schölmerich J, Rüschoff J, Kullmann F
Department of Internal Medicine I, University of Regensburg, D-93042 Regensburg, Germany.
Gut. 2003 Aug;52(8):1148-53. doi: 10.1136/gut.52.8.1148.
Colorectal epithelial cells are prone to malignant transformation. Therefore, identification of differences in gene expression in the process from normal colonic crypts to adenomas with low grade dysplasia is essential for further insights into early tumorigenesis. To achieve this goal, a novel gene expression analysis strategy, screening for expressed transcripts in small histologically defined tissue samples, was performed.
First, laser mediated microdissection was used to isolate normal and adenomatous crypts from colonic cryosections. Then, nested RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display was performed to screen mRNA populations and to generate hybridisation probes for cDNA expression arrays. After evaluation of cDNA expression arrays, differential expression was confirmed at the protein level by immunohistochemistry.
Evaluation of gene expression profiles of normal versus adenomatous colonic crypts of six different patients revealed, in general, dysregulation of up to 11% of all analysed genes (total number n=588): specifically, p21-rac1 was upregulated in four of six patients, mitogen activated protein kinase (MAPK) p38alpha in three of six patients, and interferon gamma receptor in three of six patients. Conversely, FAST kinase was found to be downregulated in three of six patients, p53 in three of six patients, and thrombospondin 2 in three of six patients.
For the first time, distinct gene expression profiles of dysplastic areas within colonic adenomas, using the combination of laser mediated microdissection with RAP-PCR and cDNA expression array, were shown. In these samples, upregulation of proliferation associated genes (ras-oncogene related p21-rac1 and MAPK p38alpha) as well as downregulation of apoptosis related genes (FAST kinase and p53) most likely reflects specific alterations in adenomas with low grade dysplasia. Based on upregulation of p21-rac1 and MAPK p38alpha, activation of the MAPK pathway appears to be an early event in colonic carcinogenesis.
大肠上皮细胞易于发生恶性转化。因此，识别从正常结肠隐窝到低级别发育异常腺瘤过程中的基因表达差异，对于进一步深入了解早期肿瘤发生至关重要。为实现这一目标，我们实施了一种新型基因表达分析策略，即在组织学定义的小样本组织中筛选表达的转录本。
首先，使用激光介导显微切割技术从结肠冰冻切片中分离正常和腺瘤性隐窝。然后，进行巢式RNA随机引物聚合酶链反应（RAP-PCR）差异显示，以筛选mRNA群体，并为cDNA表达阵列生成杂交探针。在评估cDNA表达阵列后，通过免疫组织化学在蛋白质水平确认差异表达。
对6例不同患者的正常与腺瘤性结肠隐窝的基因表达谱进行评估，总体上显示所有分析基因（总数n = 588）中高达11%的基因表达失调：具体而言，p21-rac1在6例患者中的4例中上调，丝裂原活化蛋白激酶（MAPK）p38α在6例患者中的3例中上调，干扰素γ受体在6例患者中的3例中上调。相反，FAST激酶在6例患者中的3例中下调，p53在6例患者中的3例中下调，血小板反应蛋白2在6例患者中的3例中下调。
首次展示了结合激光介导显微切割与RAP-PCR及cDNA表达阵列技术，所获得的结肠腺瘤内发育异常区域独特的基因表达谱。在这些样本中，增殖相关基因（与癌基因ras相关的p21-rac1和MAPK p38α）的上调以及凋亡相关基因（FAST激酶和p53）的下调，很可能反映了低级别发育异常腺瘤中的特定改变。基于p21-rac1和MAPK p38α的上调，MAPK通路的激活似乎是结肠癌发生过程中的早期事件。
