MEN1缺失通过诱导DNA损伤累积抑制乳腺癌细胞的恶性表型  

Loss of MEN1 suppresses the malignant phenotype of breast cancer cells by inducing accumulation of DNA damage

作　　者：梁黎 杨云巧 朱佳美 潘婷 周润蕾 邓英蕾 陈腾祥 郭兵[1,3] 李海洋 金帮明[1,2,3] 毛大华 LIANG Li;YANG Yun-qiao;ZHU Jia-mei;PAN Ting;ZHOU Run-lei;DEN Ying-lei;CHEN Teng-xiang;GUO Bing;LI Hai-yang;JIN Bang-ming;MAO Da-hua(Department of Physiology,School of Basic Medicine,Guizhou Medical University,Guiyang 550502,China;Department of Surgery,The Affiliated Hospital of Guizhou Medical University,Guiyang 550502,China;Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases,Guizhou Medical University,Guiyang 550502,China;The Wudang Affiliated Hospital of Guizhou Medical University,Guiyang 550502,China)

机构地区：[1]贵州医科大学基础医学院,贵州贵阳550502 [2]贵州医科大学附属医院肝胆外科,贵州贵阳550502 [3]贵州医科大学贵州省常见慢性病发病机制和药物研究重点实验室,贵州贵阳550502 [4]贵州医科大学附属乌当医院,贵州贵阳550502

出　　处：《中国病理生理杂志》2022年第4期616-625,共10页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82002999);贵州省科技计划项目(黔科合基础-ZK[2021]一般447);贵州省普通高等学校青年科技人才成长项目(黔教合KY字[2021]145);贵州医科大学国家自然科学基金培育项目(No.N19NSP033)。

摘　　要：目的:探讨多发性内分泌肿瘤1型综合征(multiple endocrine neoplasia type 1,MEN1)编码基因MEN1在乳腺癌细胞恶性表型调控中的作用及分子机制。方法:TCGA数据库分析乳腺癌组织(n=1097)和正常乳腺组织(n=114)中MEN1基因的mRNA表达水平;收集临床乳腺癌组织标本(n=13)和癌旁组织(n=4),采用RT-qPCR和免疫组化法检测其中MEN1基因的mRNA水平和menin蛋白的表达;CRISPR/Cas9技术构建MEN1基因敲除的MDA-MB-231(MDA-MB-231/KO)细胞,细胞经menin-MLL抑制剂MI-3单独或联合甲基硝基亚硝基胍(N-meth⁃yl-N'-nitro-N-nitrosoguanidine,MNNG)、阿霉素(doxorubicin,DOX)、紫杉醇(paclitaxel,TAX)、他莫昔芬(tamoxifen,TAM)作用后运用CCK-8、克隆形成实验、EdU染色及流式细胞术分别检测细胞增殖和细胞凋亡,免疫荧光染色法检测细胞γH2AX和53BP1的表达,Western blot法检测细胞γH2AX、RAD51、BRCA1、53BP1、KU70和KU80的蛋白表达。结果:TCGA数据库分析结果显示,与正常乳腺组织比较,MEN1基因在乳腺癌组织中的表达量显著增高(P<0.05);免疫组化和RT-qPCR结果显示,与癌旁组织比较,menin的蛋白水平和MEN1的mRNA水平显著升高(P<0.05);MEN1基因敲除或MI-3作用显著抑制乳腺癌细胞的增殖,诱导细胞凋亡,增强乳腺癌细胞对MNNG的敏感性(P<0.05);免疫荧光结果显示,MEN1基因敲除显著增强MDA-MB-231细胞核中γH2AX的染色强度而降低53BP1的染色强度(P<0.05);Western blot结果显示,MEN1基因敲除显著抑制MDA-MB-231细胞中RAD51、BRCA1及53BP1的蛋白表达,而促进KU70和KU80蛋白的表达(P<0.05)。结论:MEN1基因是乳腺癌诱导因子,MEN1缺失可抑制同源重组修复而代偿性激活非同源末端连接,引起乳腺癌细胞中DNA损伤累积,增强乳腺癌细胞对DNA损伤诱导剂的敏感性。AIM:To explore the molecular mechanisms of MEN1 gene regulates the malignant phenotype of breast cancer cells.METHODS:TCGA database analyzes the mRNA expression level of MEN1 gene in breast cancer tissues and normal breast tissues.Clinical breast cancer tissues and adjacent tissues were collected,RT-qPCR and immunohistochemistry(IHC)were used to detect the expression of MEN1 mRNA and menin protein.The knockout of MEN1 gene in MDA-MB-231 cells,named as MDA-MB-231/KO cell,was accomplished using CRISPR/Cas9 technology.After the cells were treated with menin-MLL inhibitor 3(MI-3)alone or combination with N-methyl-N’-nitro-N-nitrosoguanidine(MNNG),doxorubicin(DOX),paclitaxel(TAX)or tamoxifen(TAM)and the proliferation and apoptosis were measured by using CCK-8 method,clone formation experiment,EdU staining and flow cytometry.The expression ofγH2AX and P53-binding protein 1(53BP1)was detected by immunofluorescence.The expressions ofγH2AX,RAD51,BRCA1,53BP1,KU70 and KU80 proteins were detected by Western blot.RESULTS:The results from TCGA database analysis showed that the expression of MEN1 in breast cancer tissues was significantly higher compared with normal breast tissue(P<0.05).The results of IHC and RT-qPCR showed that the levels of menin protein and MEN1 mRNA in breast cancer tissues were significantly increased compared with adjacent tissues(P<0.05).Knockout of MEN1 gene or MI-3 treatment significantly inhibits the proliferation of breast cancer cells,induces apoptosis,and enhances the sensitivity of breast cancer cells to MNNG(P<0.05).Immunofluorescence results show that knockout of MEN1 gene increases the staining ofγH2AX and reduces the staining of 53BP1 in the nucleus of MDA-MB-231 cells(P<0.05).Western blot results show that knockout of MEN1 gene obvious inhibits the expression of RAD51,BRCA1 and 53BP1 protein,and promotes the expression of KU70 and KU80 protein(P<0.05).CONCLUSION:MEN1gene is a tumor promoter in breast cancer.Loss of MEN1 inhibits homologous recombination repair and compensatively activat

关 键 词：MEN1基因 乳腺癌 DNA损伤 同源重组修复 非同源末端连接 

分 类 号：R737.9[医药卫生—肿瘤] R730.23[医药卫生—临床医学]