miRNA-210通过HIF-1/Notch1信号通路调控乏氧乳腺癌干细胞辐射抗拒的作用  

Effects of miRNA-210 on Radioresistance of Hypoxic Breast Cancer Stem Cell via HIF-1/Notch Pathway

作　　者：齐亚莉 吴嘉慧 李忠起 陈子瑜[4] Qi Yali;Wu Jiahui;Li Zhongqi;Chen Ziyu(Department of Epidemiology,School of Public Health,Jilin Medical University,Jilin 132013;Tianjin TEDA Hospital,Tianjin 300457;Jilin International Travel Health Care Center,Changchun 130062;4731 Hospital of China Aerospace Science and Industry Corporation,Beijing 100074)

机构地区：[1]吉林医药学院公共卫生学院流行病学教研室,吉林132013 [2]天津泰达医院,天津300457 [3]吉林国际旅行卫生保健中心,长春130062 [4]中国航天科工集团七三一医院,北京100047

出　　处：《国际老年医学杂志》2021年第6期325-330,共6页International Journal of Geriatrics

基　　金：吉林省教育厅“十三五”科学研究规划项目资助(JJKH20190660KJ);吉林市科技创新发展计划项目(201830833);吉林医药学院博士科研启动基金项目(JYBS2021024LK)。

摘　　要：目的探讨miRNA-210对乏氧乳腺癌干细胞辐射抗拒的作用以及相关的分子机制。方法实验分为MCF-7/Stem、MCF-7/Stem+agomir、MCF-7/Stem+antagomir、MCF-7/Stem+H+agomir、MCF-7/Stem+H+antagomir、MCF-7/Stem+H+agomir+8 Gy和MCF-7/Stem+H+antagomir+8 Gy组。利用悬浮球法培养乳腺癌干细胞,命名为MCF-7/Stem,利用氯化钴(CoCl_(2))进行化学乏氧(hypoxia,H),转染miRNA-210的激动剂(agomir)和拮抗剂(antagomir)后,并给予8 Gy X射线照射。采用实时定量PCR法检测miRNA-210的相对表达;采用2’,7’-二氯荧光黄双乙酸盐(DCFHDA)染色,流式细胞术检测活性氧(ROS)水平;采用AnnexinⅤ-FITC/PI双染检测细胞凋亡率;采用CCK-8检测细胞增殖变化;采用Western blotting法检测HIF-1α和Notch1蛋白的表达。结果MCF-7/Stem经过乏氧后,ROS水平明显降低,并且miRNA-210表达明显增加(P<0.05)。加入miRNA-210的agomir和antagomir后4 h、12 h、24 h和48 h时,与MCF-7/Stem组相比,MCF-7/Stem+agomir组和MCF-7/Stem+antagomir组细胞吸光度A_(450)值变化不明显,而MCF-7/Stem+H+antagomir组、MCF-7/Stem+H+agomir+8 Gy组和MCF-7/Stem+H+antagomir+8 Gy组细胞吸光度A_(450)值明显降低(P<0.05),且MCF-7/Stem+H+antagomir+8 Gy组细胞吸光度A_(450)值降低得更明显。MCF-7/Stem细胞经过乏氧、转染和辐射后24 h,细胞凋亡率的变化与增殖具有相似的规律,与MCF-7/Stem组比较,MCF-7/Stem+H+antagomir+8 Gy组凋亡率增加最明显(P<0.05)。Western blotting结果显示,加入miRNA-210 antagomir后HIF-1α和Notch1蛋白表达变化不明显,而乏氧和辐射后二者的表达均增加。结论miRNA-210可以调控乏氧乳腺癌干细胞辐射抗拒,可能涉及到HIF-1/Notch1信号通路的调控。Objective To explore the effects of miRNA-210 on radioresistance of hypoxic breast cancer cells and the molecular mechanisms.Methods The cells were divided into the following groups:MCF-7/Stem,MCF-7/Stem+agomir,MCF-7/Stem+antagomir,MCF-7/Stem+H+agomir,MCF-7/Stem+H+antagomir,MCF-7/Stem+H+agomir+8 Gy,and MCF-7/Stem+H+antagomir+8 Gy.Breast cancer stem cells were obtained using suspension culture,named MCF-7/Stem.Hypoxia(H)was induced with CoCl_(2).The cells were transfected with miRNA-210 agomir or antagomir and were irradiated by 8 Gy X-ray.The relative expression of miRNA-210 was detected by quantitative real-time PCR(q RT-PCR).Reactive oxygen species(ROS)level and apoptotic rate were measured by flow cytometry.Cell proliferation was examined by CCK8 kit.HIF-1αand Notch1 protein expression were detected by western blotting.Results After hypoxia,ROS level was decreased and miRNA-210 expression was increased in MCF-7/Stem(P<0.05).At 4 h,12 h,24 h,and 48 h after transfection with miRNA-210 agomir or antagomir,compared with MCF-7/Stem,there was no change in the absorbance(A_(450))of MCF-7/Stem+agomir and MCF-7/Stem+antagomir.However,the absorbance(A_(450))of MCF-7/Stem+H+antagomir,MCF-7/Stem+H+agomir+8 Gy and MCF-7/Stem+H+antagomir+8 Gy were decreased significantly(P<0.05),and the decrease was most significant in MCF-7/Stem+H+antagomir+8 Gy.In addition,at 24 h after hypoxia,transfection and radiation,the change of apoptotic rate showed a similar trend with proliferation.Compared with MCF-7/Stem,the apoptotic rate in MCF-7/Stem+H+antagomir+8 Gy was increased significantly(P<0.05).Western blotting showed that the expression of HIF-1αand Notch1 protein did not change significantly after transfection with miRNA-210 antagomir,but they were increased after hypoxia and radiation.Conclusion MiRNA-210 may regulate radioresistance of hypoxic breast cancer stem cells,which may involve HIF-1/Notch1 signaling pathway.

关 键 词：miRNA-210 乏氧 乳腺癌干细胞 辐射 凋亡 

分 类 号：R737.9[医药卫生—肿瘤]