胃癌细胞中Ras基因的表达、突变分析及新剪接型Kras△E2的发现

Ras Gene Expression, Mutation Analysis and The Discovery of New Alternatively Spliced Isoform Kras△E2 in Gastric Cancer Cells

目的:传统Ras家族由Kras,Hras和Nras基因组成,这类基因的点突变经常在人类肿瘤中发现,突变热点位于12,13,61位密码子。ERas基因是2003年在鼠胚胎干(ES)细胞中发现的,其cDNA编码的蛋白与Kras,Hras和Nras分别有46%,43%和47%的相似性,故属于新的Ras家族成员,近几年发现ERas基因的表达与胃癌密切相关,而传统Ras基因在胃癌细胞中的表达及突变情况系统报道较少,本文旨在研究传统Ras基因Kras,Hras,Nras及其家族新成员ERas基因在胃癌细胞中的表达和突变情况。方法:选用7株不同来源不同分化程度的胃癌细胞系,利用RT-PCR及real-timePCR检测Ras基因在这些胃癌细胞系中的表达,并通过测序对传统Ras基因突变热点12,13,61位密码子及ERas基因全长进行突变分析。结果:①Ras基因在这些胃癌细胞系中均有不同程度的表达,其中Hras和Nras基因在各株细胞中表达水平均一,而Kras和ERas基因则呈差异性表达;②在这些胃癌细胞中传统Ras基因突变热点12,13,61位密码子不存在突变,ERas基因全长亦未检测到突变.③发现Kras基因一新的剪接型,特点为第一、三外显子直接拼接,缺失第二外显子,命名为Kras△E2。结论:与在其他肿瘤中不同,传统Ras基因在胃癌细胞中不存在突变热点,家族新成员ERas基因全长亦无突变,在国际上首次报道新剪接型Kras△E2,从而得出创新性结论:Ras基因家族在胃癌细胞中并不是通过热点突变导致持续活化而致癌,而可能是通过ERas基因表达量的调节或形成新的剪接型Kras△E2而致癌。另外,Kras基因是一被受国际关注的肿瘤基因,新剪接型的发现可能会对Kras基因致癌机制产生新的认识,意义重大。

Objective： The traditional Ras family consist of Kras,Hras and Nras gene, the point mutations of these genes are often found in tumors and mutations at hot-spot codons 12,13,61.ERas gene is found in mouse embryonic stem（ES） cells, the cDNA encodes a protein with 43%, 46% and 47% identity to liras, Kras and Nras respectively, therefore ERas gene is a new member of the Ras protein family. In recent years, we found that the expression of ERas gene is closely related to gastric cancer, while the expression and mutations in gastric cancer cells of other Ras genes are reported less. The purpose of this study is to analyze the expression and mutation of conventional Ras family members, KRas, HRas, NRas, and a novel family member, ERas, in gastric carcinoma （GC） strains. Methods： In this experiment, the Ras expression levels and full-length ERas transcript in 7GC strains from different sources with different differentiation degrees were determined using real-time PCR and RT-PCR. Conventional Ras gene point mutation in codon 12, 13, 61 and full-length ERas mutation were also analyzed by sequencing. Results： （1）Ras oncogenes express in all GC strains with different levels, in which HRas and NRas express similarly, but KRas and ERas express differentially. （2）ln these GC strains, the point mutation of Ras oncogenes in codon 12, 13, 61 do not exist, and no mutation is found in full-length ERas. （3）Found a new alternatively spliced isoform of KRas named KRas△E2 characterized by the first and third exons splicing directly with the second exon losing. Conclusion： Unlike Ras in other tumors, the traditional Ras genes exist no hot-spot mutations and the full length of ERas gene exist no mutations, besides the new alternatively spliced isoform is reported originally in the world, we concluded that the Ras oncogenes family, in GC cells, acquire activation of carcinogenesis probably through regulating expression level of ERas, a constitutively active Ras protein, or formation the new alternatively spliced isoform KRas △E2 rather than by point mutation. Meanwhile, Kras gene is an oncogene that attracted much attention in the internation, the found of the new alternatively spliced isoform might be bring new understanding of the carcinogenesis of Kras gene and is of great significance.