摘要
目的探讨BRCA2通过调控PI3K/AKT通路上调ATM的表达,影响乳腺癌细胞生长、迁移和侵袭。方法运用PCR技术扩增人乳腺BRCA2基因,将其克隆至真核表达载体pCDNA3.1-coGFP中,酶切和测序验证后转染至乳腺癌BT474细胞中。采用Western blot检测实验组和对照组细胞中BRCA2、ATM、Rad51、PALB2蛋白的表达,采用免疫组化分析人乳腺癌组织中BRCA2和ATM蛋白的表达,采用CCK8法和流式细胞仪分别测定各组细胞生长和凋亡变化,采用划痕实验和Transwell实验分析各组细胞迁移侵袭能力。结果从cDNA-BRCA2克隆中扩增出目的片段,并成功克隆至pCDNA3.1-coGFP载体上,经测序与目的序列完全一致;转染乳腺癌BT474细胞后BRCA2基因成功过表达;与空白和阴性对照组相比,实验组细胞生长缓慢,凋亡率增加,迁移侵袭能力降低,细胞中BRCA2、ATM、Rad51、PALB2蛋白的表达水平也明显升高(P<0.05);乳腺癌组织中BRCA2和ATM蛋白表达呈强阳性。结论过表达BRCA2基因可抑制人乳腺癌BT474细胞增殖、迁移和侵袭。BRCA2的作用机制可能与PI3K/AKT通路的失活和ATM基因的上调有关。为进一步探讨BRCA2基因在乳腺癌发生、发展过程中的作用及靶向治疗提供了基础证据。
Objective To explore the effect and mechanism of BRCA2gene up-regulating the expression of ATM protein by regulating PI3K/AKT pathway,thereby affecting breast cancer cell proliferation,invasion and migration.Methods The human BRCA2gene was amplified by PCR technology,cloned into the eukaryotic expression vector pCDNA3.1-coGFP,digested with enzyme digestion and sequencing,and then transfected into breast cancer BT474 cells.Western blot was used to detect the expression of BRCA2,ATM,Rad51,and PALB2proteins in cells of experimental group and control group.Immunohistochemistry detected the positive expression of BRCA2and ATM proteins in human breast cancer.CCK8assays and Flow cytometry were used to determine the growth and apoptosis of cells in each group.Wound-Healing Assay and Transwell assay were used to analyze the cell migration and invasion ability of each group.Results The target fragment was amplified from the cDNA-BRCA2clone and successfully cloned into the pCDNA3.1-coGFP vector.The sequencing was completely consistent with the target sequence;the BRCA2gene was successfully overexpressed after transfection into breast cancer BT474cells;Compared with the blank and negative control groups,the cells in the experimental group grew slowly,the apoptosis rate increased,the migration and invasion ability decreased,and the expression levels of BRCA2,ATM,Rad51,and PALB2protein in the cells also increased significantly(P<0.05).The expression of BRCA2and ATM proteins in breast cancer tissues is strongly positive.Conclusion Overexpression of BRCA2gene can inhibit the proliferation,migration and invasion of human breast cancer BT474 cells.The mechanism of action of BRCA2may be related to the inactivation of PI3K/AKT pathway and the up-regulation of ATM gene.This study provides basic evidence to further explore the role of BRCA2gene in the development and target treatment of breast cancer.
作者
梅楠
赵妮
陈旭阳
阮稳稳
宜烨
李春丽
MEI Nan;ZHAO Ni;CHEN Xu-yang;无(Department of Medical Oncology,The First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,China;Department of Medical Oncology,Shangluo Central Hospital,Shangluo 726000,China;Department of Medical Oncology,People’s Hospital of Ankang,Ankang 725000,China)
出处
《中国实验诊断学》
2021年第5期756-764,共9页
Chinese Journal of Laboratory Diagnosis
基金
西安交通大学第一附属医院临床科研项目(项目编号:1136900081)
