关键词: 遗传性乳腺癌, 上皮钙黏蛋白编码基因, CRISPR/Cas9, 非同源末端连接, 流式细胞术

Abstract: Background and purpose: Breast cancer is one of the most common malignant tumors in women. The discovery of a mutation gene in breast cancer may help to prevent and treat breast cancer. This article aimed to study the effect of E-cadherin gene (CDH1) and its 1018 site mutant in a DNA damage repair model. Methods: Detection of blood DNA samples from patients with breast cancer and their families revealed a missense mutation c.1018A>G (p.Thr340Ala) at the site 1018 of CDH1 gene which was suspected to be a pathogenic mutation. The CDH1 gene knockout MDA-MB-231 breast cancer cell lines were constructed. The constructed CDH1-1018 site mutaion plasmid and the non-homologous end joining (NHEJ) reporting system were transfected into cells. Cell proliferation was detected by MTT assay. Fluorescence rate of NHEJ reporting system was detected by flow cytometry. Western blot assay was used for molecular verification of key proteins related to breast cancer and NHEJ repair pathway. Results: The CDH1 gene knockout MDA-MB-231 breast cancer cell lines were established using CRISPR/Cas9. After i-sceⅠ enzyme digestion, NHEJ reporter system that transfected with CDH1-1018 site mutaion plasmid showed significantly reduced efficiency. Western blot assay confirmed that the mutation inhibited the expression of important proteins in NHEJ pathway and breast cancer related proteins. MTT assay indicated that cell proliferation efficiency decreased after transfection of CDH1-1018 site mutation plasmid. Conclusion: CDH1 gene 1018 site mutation has an inhibitory effect on the NHEJ repair pathway, and its mechanism may be related to cell adhesion and tissue movement ability, thus inhibiting the recruitment process of relevant proteins and reducing the expression.

Key words: Hereditary breast cancer, E-cadherin gene, CRISPR/Cas9, Non-homologous end joining, Flow cytometry