靶向沉默Notch1基因对人非小细胞肺癌干细胞增殖和凋亡的影响  

Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells

作　　者：刘恒铫 牟艳玲[2] 王燕[2] 王福文[2] 赵国丽 王兆朋[3] 周淑萍 蔡海波[4] 张月英[3] Liu Hengyao;Mu Yanling;Wang Yan;Wang Fuwen;Zhao Guoli;Wang Zhaopeng;Zhou Shuping;Cai Haibo;Zhang Yueying(School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan 250200, China;Institute of Materia Medica, Shandong Academy of Medical Sciences, Key Laboratory for Biotech-Drugs of National Health and Family Planning Commission, Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Jinan 250062, China;Laboratory of Pathology and Pathophysiology, Institute of Basic Medical Sciences, Shandong Academy of Medical Sciences, Jinan 250062, China;Department of Thoracic Surgery, Jining No.1 People′s Hospital, Jining 272011, China)

机构地区：[1]济南大学山东省医学科学院医学与生命科学学院,济南250200 [2]山东省医学科学院药物研究所国家卫生和计划生育委员会生物技术药物重点实验室山东省罕少见病重点实验室,济南250062 [3]山东省医学科学院基础医学研究所病理学与病理生理学研究室,济南250062 [4]济宁市第一人民医院胸外科,272011

出　　处：《国际肿瘤学杂志》2019年第2期65-71,共7页Journal of International Oncology

基　　金：国家自然科学基金(81403150);山东省自然科学基金(ZR2014HL064);山东省中医药科技发展计划(2015-325);山东省医学科学院医药卫生科技创新工程.

摘　　要：目的研究靶向沉默Notch1基因对人非小细胞肺癌干细胞增殖和凋亡的影响。方法选取人肺癌A549细胞和SPC-A-1细胞,分为空白对照组、Nc-shRNA组和Notch1-shRNA组,Nc-shRNA组为阴性对照RNAi慢病毒组,Notch1-shRNA组为Notch1抑制RNAi慢病毒组。采用慢病毒介导的shRNA干扰技术靶向沉默Notch1基因,实时定量聚合酶链反应(qRT-PCR)和Western blotting验证Notch1基因的沉默效果;采用四甲基偶氮唑蓝(MTT)法和成球实验检测细胞增殖能力;采用Annexin V/7-AAD 双染法检测细胞凋亡情况;采用Western blotting检测增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2(Bcl-2)以及Notch1下游基因Hes-1的表达情况。结果 qRT-PCR结果显示A549细胞和SPC-A-1细胞中空白对照组、Nc-shRNA组和Notch1-shRNA组Notch1相对表达量分别为1.000±0.000、0.937±0.025、0.490±0.036和1.000±0.000、1.077±0.070、0.373±0.038,差异均具有统计学意义(F=359.707,P<0.001;F=210.455,P<0.001),进一步两两比较,两种细胞中Notch1-shRNA组Notch1相对表达量明显低于Nc-shRNA组(均P<0.05);Western blotting检测A549细胞和SPC-A-1细胞中Notch1蛋白表达的结果与mRNA结果一致。MTT实验显示A549细胞球空白对照组、Nc-shRNA组和Notch1-shRNA组在24 hA值分别为0.209±0.005、0.219±0.009、0.159±0.006,48 h A值分别为0.293±0.004、0.302±0.004、0.205±0.005,72 h A值分别为0.450±0.003、0.430±0.012、0.348±0.017,差异均具有统计学意义(F=79.487,P<0.001;F=508.664,P<0.001;F=57.156,P<0.001),进一步两两比较,24、48、72 h Notch1-shRNA组增殖能力明显低于Nc-shRNA组(均P<0.05);SPC-A-1细胞球空白对照组、Nc-shRNA组和Notch1-shRNA组在48 h A值为0.438±0.022、0.412±0.015、0.364±0.010,72 h A值为0.540±0.016、0.519±0.009、0.438±0.019,差异均具有统计学意义(F=15.667,P=0.004;F=37.299,P<0.001),进一步两两比较,48、72 h Notch1-shRNA组增殖能力明显低于Nc-shRNA组(均P<0.05)。空白对照组、Nc-shRNA组和Notch1-shRNA组A549细胞成�Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells. Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group, Nc-shRNA group and Notch1-shRNA group. The Nc-shRNA group was a negative control RNAi lentivirus group, and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group. The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1. The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay. Apoptosis was detected by Annexin V/7-AAD double staining. Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1. Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group, Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000±0.000, 0.937±0.025, 0.490±0.036 and 1.000±0.000, 1.077±0.070, 0.373±0.038, with statistically significant differences (F=359.707, P<0.001;F=210.455, P<0.001), further paired comparison, the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P<0.05). Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results. MTT assay showed that the 24 h A values of A549 cells in control group, Nc-shRNA group and Notch1-shRNA group were 0.209±0.005, 0.219±0.009, 0.159±0.006, 48 h A values were 0.293±0.004, 0.302±0.004, 0.205±0.005, 72 h A values were 0.450±0.003, 0.430±0.012, 0.348±0.017, with statistically significant differences (F=79.487, P<0.001;F=508.664, P<0.001;F=57.156, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA g

关 键 词：受体 Notch1 肿瘤干细胞 癌 非小细胞肺 细胞增殖 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]