摘要
目的探讨沉默BLM DNA解旋酶基因对三阴性乳腺癌MDA-MB-468细胞增殖和凋亡的影响。方法利用数据库分析BLM DNA解旋酶在乳腺癌组织中的表达情况及后评估价值,免疫组织化学技术检测三阴性乳腺癌、乳腺纤维腺瘤和癌旁组织中BLM DNA解旋酶表达,Western blot检测MCF-10A细胞、MCF-7细胞、MDA-MB-468细胞中BLM DNA解旋酶蛋白表达水平;合成siRNA序列沉默MDA-MB-468细胞中BLM DNA解旋酶基因的表达,选取沉默效率最高的siRNA-BLM 3序列构建shRNA干扰载体慢病毒;将慢病毒感染MDA-MB-468细胞分为shRNA-NC组和shRNA-BLM组,MTT、平板克隆形成实验检测细胞增殖能力,流式细胞术检测细胞凋亡情况,单细胞凝胶电泳技术检测与DNA损伤相关的细胞尾部DNA含量,免疫细胞化学检测DNA损伤标志物γ-H2AX表达情况,Western blot检测凋亡相关蛋白及DNA损伤修复蛋白表达情况。结果生物信息学分析显示BLM DNA解旋酶在乳腺癌组织中高表达,且与乳腺癌患者不良预后密切相关;三阴性乳腺癌组织BLM DNA解旋酶表达水平高于乳腺纤维腺瘤和癌旁组织(P<0.001);与MCF-10A细胞、MCF-7细胞比较,MDA-MB-468细胞中BLM DNA解旋酶蛋白表达水平升高(P<0.01);与未转染组比较,siRNA-BLM 3组BLM DNA解旋酶蛋白表达下调(P<0.05);与shRNA-NC组比较,shRNA-BLM组细胞增殖能力被抑制、凋亡率升高、尾部DNA含量增加、γ-H2AX阳性细胞比例升高(P<0.01)、凋亡相关蛋白Bax、cleaved Caspase-3、cleaved Caspase-8表达上调,Bcl-2及DNA损伤修复蛋白BRCA1、Rad51、BLM表达下调(P<0.05)。结论沉默BLM DNA解旋酶基因表达抑制三阴性乳腺癌细胞增殖,促进细胞凋亡,凋亡机制可能与细胞DNA损伤增加,DNA损伤修复相关蛋白受抑制有关。
Objective To investigate the effects of silencingBLMDNA helicase expression on proliferation and apoptosis of MDA-MB-468 triple-negative breast cancer cells.Methods The database was utilized to analyze BLM DNA helicase expression level in breast cancer tissues and its prognostic value.Immunohistochemistry(IHC) was applied to detect BLM DNA helicase expression in triple-negative breast cancer tissues, breast fibroadenoma and paracancerous tissues.Western blot was used to detect the protein expression of BLM DNA helicase in MCF-10A, MCF-7, and MDA-MB-468cell lines.Synthesized siRNA sequences againstBLMDNA helicase expression were first used to verify their silencing efficacy in MDA-MB-468 cells.siRNA-BLM 3 sequence with the highest silencing efficacy was selected to generate shRNA lentivirus vector for producing lentivirus.MDA-MB-468 cells were infected with lentivirus carrying shRNA-NC or shRNA-BLM, and further divided into shRNA-NC and shRNA-BLM groups.MTT and colony formation assays were performed to detect cell proliferation.Flow cytometry was applied to examine cellular apoptosis.Single-cell gel electrophoresis(SCGE) was used to detect DNA damage by evaluating the percentage of DNA in comet tails.Immunocytochemistry was applied to detect DNA damage-associated γ-H2AX expression.Western blot was performed to detect the expression levels of apoptosis-related proteins and DNA damage repair-related proteins.Results Bioinformatics analysis showed that BLM DNA helicase was highly expressed in breast cancer tissues and closely related to the poor prognosis of patients with breast cancer.The expression level of BLM DNA helicase in triple-negative breast cancer tissues was the highest relative to those in breast fibroadenomas and paracancerous tissues(P< 0.001).Similarly, the expression level of BLM DNA helicase in MDA-MB-468 cells was the highest relative to those in MCF-10A and MCF-7 cell lines(P< 0.01).When compared to the non-transfected group, the expression level of BLM DNA helicase in siRNA-BLM 3 group was down-regulated(P< 0.05).Cell proliferation was decreased, but cellular apoptotic rate, DNA contents in comet tails and the proportion of γ-H2AX-positive cells were increased in shRNA-BLM group when compared to shRNA-NC group(P< 0.01).The expression levels of apoptosis-related proteins such as Bax, cleaved Caspase-3 and cleaved Caspase-8 were upregulated, while the expression levels of Bcl-2, DNA damage repair-related proteins such as BRCA1, Rad51, and BLM were downregulated in shRNA-BLM group relative to shRNA-NC group(P< 0.05).Conclusion Silencing BLMDNA helicase expression inhibits the triple-negative breast cancer cell proliferation and promotes cellular apoptosis.Its mechanism may relate to the increase of cellular DNA damage and the inhibition of DNA damage repair-related proteins.
作者
黄亚兰
张望明
刘小花
余晓静
安丽君
吴介恒
杨留启
刘杰麟
HUANG Yalan;ZHANG Wangming;LIU Xiaohua;YU Xiaojing;AN Lijun;WU Jieheng;YANG Liuqi;LIU Jielin(Department of Immunology,School of Basic Medical Science,Guizhou Medical University,Guiyang 550025,Guizhou,China;State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550014,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2022年第8期869-878,共10页
Journal of Guizhou Medical University
基金
国家自然科学基金(81360349)
贵州省科技支撑项目(黔科合支撑[2020]4Y120)
贵州医科大学省部共建药用植物功效与利用国家重点实验室开放课题(FAMP202110K)。
