甲基丙烯酸环氧丙酯诱导16HBE细胞恶性转化过程中细胞凋亡相关lncRNA的筛选及功能研究  

Long non-coding RNA regulates apoptosis during glycidyl methacrylate-induced malignant transformation of 16HBE cells

作　　者：马顺鹏 王全凯[1,2] 王苗 宋佳阳 乌瀚宝栎尔 谢广云[1] 许建宁[1,2] MA Shunpeng;WANG Quankai;WANG Miao;SONG Jiayang;WUHAN Baolier;XIE Guangyun;XU Jianning(National Institute of Occupational Health and Poison Control,Chinese Center for Disease Control and Prevention,Beijing 100050;Key Laboratory of Chemical Safety and Health Chinese Center for Disease Control and Prevention,Beijing 100050,China)

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,北京100050 [2]中国疾病预防控制中心化学污染与健康安全重点实验室,北京100050

出　　处：《癌变．畸变．突变》2021年第1期1-5,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81673221)。

摘　　要：目的:筛选在甲基丙烯酸环氧丙酯(GMA)诱导人支气管上皮16HBE细胞恶性转化过程中凋亡相关lncRNA,并探讨其对细胞恶性转化的3个时期细胞凋亡的调控作用。方法:取处于对数生长期的16HBE细胞,二甲基亚砜(DMSO)作为溶剂对照,终浓度为8μg/mL的GMA为处理组,每次染毒72 h,重复染毒3次后,分别进行传代培养。收集GMA染毒组和DMSO对照组的第10、20和30代细胞,基于细胞转化表型鉴定结果,将其分为转化前、中和后期。流式细胞术检测3个时期的细胞凋亡率,lncRNA表达谱芯片筛选3个时期细胞凋亡相关差异表达lncRNAs,生物信息学数据库预测差异lncRNA可能的作用靶基因。实时荧光定量PCR验证筛选出的差异lncRNA相对表达水平。结果:与同时期DMSO对照组相比,第10代GMA染毒组的细胞凋亡率明显升高,第20代GMA染毒组的细胞凋亡率降低(均为P<0.05),第30代GMA染毒组的细胞凋亡率与同时期的DMSO对照组相比差异无统计学意义。芯片筛选结果发现,lncRNA CFLAR-AS1在第10、20和30代分别下调22%、上调244%和下调30%,其可能靶基因CFLAR的mRNA分别上调27%、下调30.6%和下调31%。lncRNA CFLAR-AS1的qPCR验证结果与芯片筛选结果一致,与同时期的DMSO对照组相比,第10、20和30代的GMA染毒组细胞分别下调42%、上调442%和下调9%,其中第10和20代细胞与对照组间的差异具有统计学意义(P<0.05)。结论:在GMA诱导16HBE恶性转化过程中,转化前期凋亡率升高,转化中期凋亡率下降,这一时期lncRNA CFLAR-AS1可能发挥抑制细胞凋亡的作用,但其作用机制还有待进一步研究。OBJECTIVE:To screen for apoptosis-related lncRNAs and to investigate their regulatory effects on glycidyl methacrylate(GMA)-induced malignant transformation of human bronchial epithelial(16HBE)cells.METHODS:The 16HBE cells in the logarithmic growth phase were exposed to dimethyl sulfoxide(DMSO)as the solvent control group,and to 8μg/mL GMA as the treatment group.Cells were subcultured after repeated exposure 3 times for 72 hours each time.Subsequently,cells at the 10th,20th and 30th generations were collected.Based on cell transformation phenotypes of the cultured cells,they were divided into pre-,middle-and late-transformation stages.Flow cytometry was used to detect expression of apoptosis and apoptosis-related lncRNAs were screened from the lncRNA microarrays of the three transformation stages.Bioinformatics database was used to find the target genes of the differentially expressed lncRNAs.Real-time quantification PCR was used to detect the relative expression of differential lncRNAs.RESULTS:Compared with the control group of the same(10th)generation,the apoptosis levels of the GMA-treated cells were significantly increased.The apoptosis levels of the 20th generation GMA-treated cells were significantly lower than the same generation control group(P<0.05)and were not significantly different from the control group of the 30th generation.The results from the lncRNA microarrays show that lncRNA CFLAR-AS1 was downregulated 22%,up-regulated 244%and down-regulated 30%in the 10 th,20th and 30th generations after GMA-treatments,suggesting that the target mRNA CFLAR was up-regulated 27%,down-regulated 30.6%and down-regulated 31%.The results from qPCR verification of lncRNA CFLAR-AS1 were consistent with the results of the lncRNA microarrays which were down-regulated 42%,up-regulated 442%and down-regulated 9%in the 10th,20th and 30th generations,respectively.The difference between the 10th and the 20th generations was statistically significant(P<0.05).CONCLUSION:In the process of malignant transformation of 16HBE from

关 键 词：甲基丙烯酸环氧丙酯 人支气管上皮细胞 细胞恶性转化 细胞凋亡 长链非编码RNA 

分 类 号：R114[医药卫生—卫生毒理学]