Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants
Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants
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DOI：
10.1136/gut.51.5.677
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发表时间：
2002-11-01
期刊：
影响因子：
24.5
通讯作者：
Zeuzem, S
中科院分区：
文献类型：
--
作者：
Brieger, A;Trojan, J;Zeuzem, S
背景：错配修复 (MMR) 基因 hMLH1 和 hMSH2 的种系突变可导致遗传性非息肉病性结直肠癌 (HNPCC)。然而，hMLH1和hMSH2突变的功能性体外分析仍然困难。目的：建立hMLH1和hMSH2突变功能表征的体外方法。方法：将hMLH1和hMSH2野生型（wt）基因和几个突变亚克隆瞬时转染到错配修复缺陷细胞系（HCT-116和LOVO）中。分别通过Hoechst染色、AlamarBlue染色、实时聚合酶链反应和western blotting分析细胞凋亡、增殖以及相互作用蛋白mRNA表达和蛋白表达的调节。结果：与野生型转染相比，转染后hMLH1和hMSH2突变体的蛋白表达显着降低。 hMLH1 和 hMSH2 相互作用蛋白 hPMS2 和 hMSH6 仅在转染各自的野生型基因后才可检测到。与此同时，hMSH2 wt 转染细胞中的 hMSH6 mRNA 水平增加。然而，hPMS2 mRNA 水平与其相互作用伙伴 hMLH1 的突变状态无关，表明存在转录后调节途径。在 hMLH1 缺陷的 HCT-116 细胞系中，任何错配修复基因的转染都不会影响细胞凋亡，而在 LOVO 细胞中补充 hMSH2 缺陷会增加细胞凋亡。相反，HCT-116的增殖活性通过与hMLH1wt互补而降低，并且在hMSH2缺陷的LOVO细胞中不受影响。结论：这些数据表明，MMR基因及其突变的细胞作用可以在简单的瞬时转染系统中评估，并显示MMR基因调节对主要细胞生长调节机制的影响。该方法适用于疑似 HNPCC 患者中观察到的 hMLH1 和 hMSH2 基因突变的功能定义。
Background: Germline mutations in the mismatch repair (MMR) genes hMLH1 and hMSH2 can cause hereditary non-polyposis colorectal cancer (HNPCC). However, the functional in vitro analysis of hMLH1 and hMSH2 mutations remains difficult.Aims: To establish an in vitro method for the functional characterisation of hMLH1 and hMSH2 mutations.Methods: hMLH1 and hMSH2 wild type (wt) genes and several mutated subclones were transiently transfected in mismatch repair deficient cell lines (HCT-116 and LOVO). Apoptosis, proliferation, and regulation of mRNA expression and protein expression of interacting proteins were analysed by Hoechst staining, AlamarBlue staining, real time polymerase chain reaction, and western blotting, respectively.Results: The protein expression of hMLH1 and hMSH2 mutants was significantly decreased after transfection compared with wild type transfections. The hMLH1 and hMSH2 interacting proteins hPMS2 and hMSH6 became detectable only after transfection of the respective wild type genes. In parallel, hMSH6 mRNA levels were increased in hMSH2 wt transfected cells. However, hPMS2 mRNA levels were independent of the mutation status of its interacting partner hMLH1, indicating a post-transcriptional regulating pathway. In the hMLH1 deficient HCT-116 cell line apoptosis was not affected by transfection of any mismatch repair gene, whereas complementation of hMSH2 deficency in LOVO cells increased apoptosis. Conversely, proliferative activity of HCT-116 was decreased by complementation with hMLH1wt and unaffected in hMSH2 deficient LOVO cells.Conclusion: These data show that the cellular role of the MMR genes and its mutations are assessable in a simple transient transfection system and show the influence of MMR gene regulation on major cell growth regulating mechanisms. This method is applicable for the functional definition of mutations in hMLH1 and hMSH2 genes observed in patients with suspected HNPCC.