Baines Antonio T, Lim Kian-Huat, Shields Janiel M, Lambert John M, Counter Christopher M, Der Channing J, Cox Adrienne D
Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Methods Enzymol. 2006;407:556-74. doi: 10.1016/S0076-6879(05)07045-X.
Cancer is a multistep genetic process that includes mutational activation of oncogenes and inactivation of tumor suppressor genes. The Ras oncogenes are the most frequently mutated oncogenes in human cancers (30%), with a high frequency associated with cancers of the lung, colon, and pancreas. Mutational activation of Ras is commonly an early event in the development of these cancers. Thus, whether mutated Ras is required for tumor maintenance and what aspects of the complex malignant phenotype might be promoted by mutated Ras are issues that remain unresolved for these and other human cancers. The recent development of interfering RNA to selectively impair expression of mutated Ras provides a powerful approach to begin to resolve these issues. In this chapter, we describe the use of retrovirus-based RNA interference approaches to study the functions of Ras and Ras effectors (Raf, RalA, RalB, and Tiam1) in the growth of pancreatic carcinoma and other human tumor cell lines. Finally, we also compare the use of constitutive and inducible shRNA expression vectors for analyses of mutant Ras function.
癌症是一个多步骤的遗传过程，包括癌基因的突变激活和肿瘤抑制基因的失活。Ras癌基因是人类癌症中最常发生突变的癌基因（30%），在肺癌、结肠癌和胰腺癌中出现频率很高。Ras的突变激活通常是这些癌症发展过程中的早期事件。因此，对于这些以及其他人类癌症来说，突变的Ras是否是肿瘤维持所必需的，以及突变的Ras可能会促进复杂恶性表型的哪些方面，这些问题仍未得到解决。最近干扰RNA的发展，可选择性地损害突变Ras的表达，为开始解决这些问题提供了一种有力的方法。在本章中，我们描述了基于逆转录病毒的RNA干扰方法在研究Ras及其效应分子（Raf、RalA、RalB和Tiam1）在胰腺癌和其他人类肿瘤细胞系生长中的功能方面的应用。最后，我们还比较了组成型和诱导型短发夹RNA（shRNA）表达载体在分析突变Ras功能方面的应用。
