分化拮抗非蛋白编码RNA调控乳腺癌细胞增殖的机制研究  

Mechanism of differentiation antagonizing non-protein-coding RNA regulating proliferation of breast cancer cells

作　　者：韩丽飞 吕建鑫[1] 王宝偲[1] 曹欣华 马骁 胡浩霖[1] 张亚男[1] Han Lifei;Lyu Jianxin;Wang Baocai;Cao Xinhua;Ma Xiao;Hu Haolin;Zhang Yanan(Breast Disease Center,Zhongda Hospital,Southeast University,Nanjing 210009,China)

机构地区：[1]东南大学附属中大医院乳腺中心,南京210009

出　　处：《中华乳腺病杂志（电子版）》2023年第1期21-29,共9页Chinese Journal of Breast Disease(Electronic Edition)

基　　金：国家自然科学基金青年科学基金资助项目(81502287)。

摘　　要：目的研究分化拮抗非蛋白编码RNA(DANCR)在乳腺癌中可能的作用机制。方法使用定量RT-PCR检测DANCR在2017年1月至2018年1月东南大学附属中大医院收治的26例未行新辅助治疗的乳腺癌患者癌组织和癌旁正常组织中以及正常乳腺上皮细胞(MCF-10A)和不同乳腺癌细胞系(MCF-7、MDA-MB-231、SK-BR-3)中的表达水平。分别用阴性对照、短发夹RNA(shRNA)慢病毒载体转染MDA-MB-231细胞,得到阴性对照组、shRNA(381)和shRNA(766)3组细胞。分别采用MTT、Transwell实验、流式细胞技术检测3组细胞增殖、迁移、凋亡和细胞周期的差别。采用Western blot检测3组细胞中细胞周期蛋白D1(cyclin D1)和周期蛋白依赖性激酶4(CDK4)表达的变化。乳腺癌患者癌组织和癌旁正常组织中DANCR表达量的组间比较采用配对t检验。以DANCR表达中位数1.344作为截止值,将患者分为DANCR高表达组和DANCR低表达组,采用Fisher精确概率法比较2组患者临床病理特征的差别。4种细胞系中DANCR表达量的组间比较、3组MDA-MB-231细胞存活率、细胞迁移数、凋亡细胞比例、细胞周期数比例和cyclin D1和CDK4蛋白表达量的比较均采用单因素方差分析,进一步两两比较采用LSD法。结果(1)DANCR在乳腺癌组织中的表达量高于癌旁正常组织,差异具有统计学意义[2.02±0.33比1.07±0.03,t=-2.875,P=0.008]。26例患者中DANCR高表达组13例,DANCR低表达组13例,2组患者的TNM分期、ER表达比较,差异具有统计学意义(P=0.002、0.047)。(2)乳腺癌细胞系MCF-7、SK-BR-3、MDA-MB-231和正常乳腺上皮细胞系MCF-10A中DANCR的表达量分别为1.46±0.23、2.33±0.22、2.14±0.08和1.00±0.09,组间比较差异具有统计学意义(F=35.656,P<0.001)。与正常乳腺上皮细胞MCF-10A相比,SK-BR-3细胞系和MDA-MB-231细胞系中的DANCR表达量均显著增加(P均<0.001)。(3)DANCR表达量在阴性对照组、shRNA(381)和shRNA(766)3组细胞中分别为1.00±0.07、0.08±0.01和0.28±Objective To investigate the potential mechanism of differentiation antagonizing non-protein-coding RNA(DANCR)in breast cancer cells.Methods Fresh primary breast cancer samples and paired adjacent normal tissue samples were obtained from 26 patients in the Zhongda Hospital,Southeast University from January 2017 to January 2018.None of the patients received neoadjuvant therapy.DANCR expression was detected by qRT-PCR in breast cancer tissues and adjacent normal tissue,and in some cell lines(MCF-7,MDA-MB-231,SK-BR-3 and MCF-10A).MDA-MB-231 cells were transfected with negative control lentivirus and short hairpin RNA(shRNA)lentivirus respectively to obtain three groups of cells:negative control group,shRNA(381)group and shRNA(766)group.MTT,Transwell assay and flow cytometry were used to evaluate the proliferation,migration,apoptosis and cell cycle of MDA-MB-231 cells.Western blot analysis was used to detect the expression of cyclin D1 and cyclin-dependent kinase kinase 4(CDK4)in three cell lines.Paired t test was used to compare the expression of DANCR between breast cancer tissue and adjacent normal tissue.Using the median value of DANCR expression(1.344)as a cutoff,patients were divided into two groups:high DANCR expression group and low DANCR expression group.Fisher’s exact test was used to compare the clinicopathological characteristics between two groups.The expression level of DANCR among four cell lines,cell survival rate,migrated cell number,apoptotic rate,percentage of cells in different cell cycle and the expression levels of cyclin D1 and CDK4 in MDA-MB-231 cells among three groups were compared by one-way analysis of variance.The LSD method was used for pairwise comparison.Results(1)The expression of DANCR in breast cancer tissue was significantly higher than that in adjacent normal tissue(2.02±0.33 vs 1.07±0.03,t=-2.875,P=0.008).Among the 26 patients,13 patients showed DANCR high expression and 13 patients showed low expression.There was a significant difference in TNM stage and ER expression betwee

关 键 词：乳腺肿瘤 RNA 长链非编码 增殖 迁移 细胞周期 

分 类 号：R737.9[医药卫生—肿瘤]