人胃癌Ha-ras基因及上游区片段甲基化对转化效率及表达的影响

Effects of Methylation on the Transforming Activity and Expression of Oncogene Ha-ras

本文将克隆于pBR322的人胃癌Ha-ras基因(PGC6.6)和带有上游区片段的Ha-ras基因(PGC9.1)的CC~*GG位点甲基化后,转化NIH3T3细胞。发现pGC6.6甲基化与非甲基化对转化效率无明显影响,而pGC9.1甲基化后转化效率明显低于非甲基化pGC9.1者,甲基化/非甲基化pGC9.1的转化效率均明显高于甲基化/非甲基化pGC6.6者。本文又对人胃癌组织及癌旁组织DNA中Ha-ras基因的HpaⅡ、Msp Ⅰ限制性内切酶图谱作了比较,并同对比较了癌及癌旁组织中Ha-ras基因的mRNA水平,发现一例病人癌组织中Ha-ras基因的CC~*GG位点甲基化程度较癌旁组织中者低,且该例中Ha-ras基因表达水平在癌组织中明显地高。这些结果,结合我们以前的研究表明:在人胃Ha-ras癌基因上游区可能存在一增强子样作用的区域,对Ha-ras基因起调控作用。该上游区CC~*GG位点的甲基化能降低这种调控作用。仅Ha-ras结构基因的CC~*GG位点甲基化不足以明显影响其转化活性。在体内,Ha-ras基因甲基化水平降低可能与其达表水平升高以至诱发癌症有关。

Plasmids containing Ha-ras oncogene (pGC6.6) and Ha-ras oncogene attached with an upstream fragment(pGC9.1)were methylated in vitro with the sequence specific bacterial methyltrans ferase Hpa Ⅱ. The transforming activity of the treated-plasmids was assayed by transforming NIH3T3 cells. The trasforming efficiency of methylated pGC6.6 is the same as that of it's non-methylated counterpart, but the transforming efficiency of methylated pGC9.1 is significantly lower than that of the non-methylated pGC9.1. However, the transforming efficiency of pGC9.1, whether methylated or not is significantly higher than that of pGC6.6. In vivo study on the effect of Ha-ras oncogene methylation on the level of expression showed that in some patient with lower methylation level at CC GG site of Ha-ras gene, the expressin level of Ha-ras mRNA in the cancer tissue is higher than in the surrounding peripheral tissue. These results and our previous studies suggest that in the upstream region of Ha-ras oncogene in human gastric carcinoma a cis-acting enhancer-like element may be present, and the methylation of CC GG site in the upstream region may have a negative effect on the function of this element. Besides, there is a correlation between the lower methylation and higher expression of this oncogene in vivo.