Patek Charles E, Arends Mark J, Rose Lorraine, Luo Feijun, Walker Marion, Devenney Paul S, Berry Rachel L, Lawrence Nicola J, Ridgway Rachel A, Sansom Owen J, Hooper Martin L
Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, The University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK.
BMC Gastroenterol. 2008 Jun 13;8:24. doi: 10.1186/1471-230X-8-24.
Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastmDelta4A/tmDelta4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively.
The relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice were generated and the genotypes confirmed by PCR analysis. Survival of stocks was compared by the Mantel-Haenszel test, and tumour number and area compared by Student's t-test in outwardly healthy mice at approximately 106 and 152 days of age. DNA sequencing of codons 12, 13 and 61 was performed to confirm the intestinal tumours did not harbour a K-ras activating mutation.
The K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology.
The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations.
基因剪接改变发生在人类散发性结直肠癌（CRC）中，可能促进肿瘤进展。K-ras原癌基因编码两种剪接变体，K-ras 4A和4B，在CRC中，共同影响这两种异构体的K-ras激活突变很常见。过去的研究证实，CRC中K-ras癌基因和原癌基因的剪接均发生改变，有利于K-ras 4B。本研究利用自发发生不携带K-ras激活突变的肠道肿瘤的ApcMin/+（Min）小鼠，以及仅能表达K-ras 4B剪接变体的K-rastmDelta4A/tmDelta4A小鼠，探讨在不存在致癌等位基因的情况下，K-Ras 4A原癌蛋白是否能抑制肿瘤发展。通过这种方式，分别比较了能表达和不能表达K-ras 4A原癌蛋白的ApcMin/+、K-ras+/+小鼠与ApcMin/+、K-rastmDelta4A/tmDelta4A小鼠小肠中的肿瘤发生情况。
通过实时RT-qPCR分析定量正常小肠和小肠肿瘤中K-ras剪接变体的相对表达水平。培育近交（C57BL/6）ApcMin/+、K-ras+/+和ApcMin/+、K-rastmDelta4A/tmDelta4A小鼠，并通过PCR分析确认基因型。通过Mantel-Haenszel检验比较小鼠种群的存活率，通过Student's t检验比较约106日龄和152日龄外表健康小鼠的肿瘤数量和面积。对密码子12、13和61进行DNA测序，以确认肠道肿瘤不携带K-ras激活突变。
K-ras 4A转录本约占野生型和Min小鼠小肠中K-ras表达量的50%。Min小鼠小肠肿瘤中K-ras 4B转录本表达水平升高，但K-ras 4A转录本水平无明显变化。在来自Min和复合突变Min小鼠的27个肠道肿瘤中未检测到K-ras激活突变。K-Ras 4A缺陷不影响小鼠存活率、肿瘤数量、大小或组织病理学。
即使在缺乏K-ras激活突变的腺瘤中K-ras 4A/4B比例降低，K-Ras 4A原癌蛋白在小肠中也不表现出肿瘤抑制活性。
