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

doi:10.3969/j.issn.1004-616x.2021.01.001

癌变·畸变·突变 ›› 2021, Vol. 33 ›› Issue (1): 1-5,31.doi: 10.3969/j.issn.1004-616x.2021.01.001

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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

马顺鹏^1,2, 王全凯^1,2, 王苗^1,2, 宋佳阳¹, 乌瀚宝栎尔¹, 谢广云¹, 许建宁^1,2

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

收稿日期:2020-11-09 修回日期:2020-12-15 出版日期:2021-01-30 发布日期:2021-02-06
通讯作者: 许建宁,E-mail:jnx999@263.net E-mail:jnx999@263.net
作者简介:马顺鹏,E-mail:msp_ny@163.com。
基金资助:
国家自然科学基金（81673221）

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

MA Shunpeng^1,2, WANG Quankai^1,2, WANG Miao^1,2, SONG Jiayang¹, WUHAN Baolier¹, XIE Guangyun¹, XU Jianning^1,2

1. National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050;
2. Key Laboratory of Chemical Safety and Health Chinese Center for Disease Control and Prevention, Beijing 100050, China

Received:2020-11-09 Revised:2020-12-15 Online:2021-01-30 Published:2021-02-06

摘要/Abstract

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8 μg/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。

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

Abstract: 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 8 μg/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-, middleand 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 down-regulated 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 repeated exposure to GMA, the pre-transformation apoptosis ratio increased and the mid-transformation apoptosis ratio decreased. During this period, lncRNA CFLAR-AS1 played a role in inhibiting apoptosis but the mechanism for this function needs further study.

Key words: glycidyl methacrylate, human bronchial epithelial cells, malignant transformation, apoptosis, lncRNA

中图分类号:

R114

马顺鹏, 王全凯, 王苗, 宋佳阳, 乌瀚宝栎尔, 谢广云, 许建宁. 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[J]. 癌变·畸变·突变, 2021, 33(1): 1-5,31.

MA Shunpeng, WANG Quankai, WANG Miao, SONG Jiayang, WUHAN Baolier, XIE Guangyun, XU Jianning. Long non-coding RNA regulates apoptosis during glycidyl methacrylate-induced malignant transformation of 16HBE cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2021, 33(1): 1-5,31.

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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

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