微卫星不稳定型大肠癌中基因突变及其产物的检测

Detection of frameshift mutation and mutant gene products in the colorectal cancer with microsatellite instability

目的通过检测基因突变及其产物在微卫星不稳定型大肠癌中的表达,探讨微卫星不稳定型大肠癌发生发展的分子生物学机制。方法利用PCR、RT-PCR及蛋白印迹杂交方法,在11个大肠癌细胞株中检测微卫星稳定程度;9个蛋白质编码区域存在重复碱基序列的,与细胞生长、DNA修复及细胞凋亡相关基因中检测基因突变及其mRNA和蛋白质表达。结果检测结果发现了7个大肠癌细胞株表现高度微卫星不稳定,1个大肠癌细胞株表现低度微卫星不稳定,3个大肠癌细胞株表现微卫星稳定。与微卫星稳定的细胞株相比,微卫星不稳定型大肠癌中基因突变率显著增高。ACVRⅡ、Bax、hMSH6、hRad50、RIZ和TGFβRⅡ等6个基因突变率超过50%;ACVRⅡ、ATR、Bax、MBD4、hMSH3、RIZ和TGFβRⅡ7个基因表达突变型mRNA;而hMSH6和hRad50不表达突变型mRNA;同时hRad50的蛋白质表达与无突变的细胞株相比显著下降。结论与细胞生长,DNA修复,细胞凋亡相关基因的高度突变及其突变基因产物与微卫星不稳定型大肠癌的发生有密切相关,hRad50蛋白表达降低可能与微卫星不稳定型大肠癌的染色体不稳定有密切关系。

Purpose To examine frameshift mutations and mutant gene products in the colorectal cancer with microsatellite instability. Methods Nine genes associated with cell growth, DNA repair, and apoptosis which have mononucleotide repeated sequence in their coding region were selected in this study. Microsatellite status, frameshift mutation, and mutant gene products were examined in 11 colorectal cancer cell lines using PCR, RT-PCR, and Western blotting. Results Seven colorectal cancer cell lines showed high-microsatellite instability （MSI-H）, 1 showed low-microsatellite instability （MSI-L）, and 3 showed microsatellite stable （MSS）. Frameshift mutation ratio was significantly higher in MSI-H cell lines than in MSS cell lines; mutation ratios of ACVR Ⅱ , Bax, hMSH6, hRadSO, RIZ, and TGFβR Ⅱ were over 50% ; ACVR Ⅱ , ATR, Bax, MBD4, hMSH3, RIZ, and TGFβR Ⅱ showed mutant mRNA, while hMSH6 and hRadSO did not show mutant mRNA. hRadSO protein expression was significantly decreased in mutant cell lines compared to wild type cell lines. Conclusions High frameshift mutation and mutant mRNA expression in cell growth, DNA repcir, and apoptosis associated genes may play an important role in the tumorigenesis of colorectal cancer with microsatellite instability. Decreased expression of hRadSO may be associated with chromosomal imbalance in the colorectal cancer with microsatellite instability.