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中国癌症防治杂志

中国癌症防治杂志 ›› 2023, Vol. 15 ›› Issue (1): 11-17.doi: 10.3969/j.issn.1674?5671.2023.01.02

• 基础研究 • 上一篇    下一篇

过表达miR⁃486⁃5p增强鼻咽癌放射抗拒细胞株CNE⁃2R放射敏感性

  

  1. 广西医科大学附属肿瘤医院放疗科；柳州市工人医院/广西医科大学第四附属医院肿瘤科；广西医科大学附属武鸣医院肿瘤科
  • 出版日期:2023-02-25 发布日期:2023-03-01
  • 通讯作者: 朱小东 E-mail：zhuxdonggxmu@126.com
  • 基金资助:
    国家自然科学基金项目（81760544）；广西医科大学青年科学基金项目（GXMUYSF202229）；广西壮族自治区卫生健康委员会自筹经费科研课题（Z20200269）

Overexpressing miR⁃486⁃5p enhanced radiosensitivity of radioresistant nasopharyngeal carcinoma cell line CNE⁃2R

  • Online:2023-02-25 Published:2023-03-01

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摘要： 目的 探讨鼻咽癌放射抗拒细胞株CNE⁃2R过表达miR⁃486⁃5p后的放射增敏效应。方法 采用microRNA（miRNA）测序和RT⁃qPCR检测miR⁃486⁃5p在鼻咽癌亲本细胞CNE2和放射抗拒细胞CNE⁃2R中的表达水平。使用miR⁃486⁃5p过表达及阴性对照慢病毒液感染CNE⁃2R细胞，分别定义为CNE⁃2R⁃Mimics组和CNE⁃2R⁃NC组，利用CCK⁃8实验、平板克隆形成实验检测细胞增殖能力和经不同照射剂量（0 Gy、2 Gy、4 Gy、6 Gy、8 Gy）X射线照射后的细胞活力，Annexin V⁃APC/7⁃AAD双染法流式细胞术检测细胞凋亡情况。结果 miRNA测序和RT⁃qPCR结果均显示CNE⁃2R细胞中的miR⁃486⁃5p表达水平低于亲本细胞CNE2。CCK⁃8实验结果显示，培养48 h、72 h、96 h后，过表达miR⁃486⁃5p的CNE⁃2R细胞增殖能力较CNE⁃2R⁃NC组减弱（均P<0.05）；经2 Gy、4 Gy、6 Gy、8 Gy照射剂量照射后，CNE⁃2R⁃Mimics组存活分数低于CNE⁃2R⁃NC组（均P<0.05），其中在8 Gy照射剂量下两组存活分数差异最大（t=30.152，P<0.001）。流式细胞术实验结果显示，在未照射（0 Gy）和8 Gy照射剂量下，CNE⁃2R⁃Mimics组凋亡率均高于CNE⁃2R⁃NC组（均P<0.05），其中在8 Gy剂量照射下细胞凋亡率差异更显著（t=14.945，P<0.001）。平板克隆形成实验结果显示，在不同照射剂量照射下，CNE⁃2R⁃Mimics组克隆形成数均低于CNE⁃2R⁃NC组，其中CNE⁃2R⁃Mimics组的放射生物学参数D0、Dq和照射2 Gy时的存活分数均小于CNE⁃2R⁃NC组（均P<0.05）。结论 鼻咽癌放射抗拒细胞株CNE⁃2R过表达miR⁃486⁃5p后可能通过抑制细胞增殖和促进细胞凋亡来增加放射敏感性。

关键词: 鼻咽癌, miR-486-5p, 放射增敏, 增殖, 凋亡

Abstract: Objective To investigate the radiosensitivity effect of radioresistant nasopharyngeal carcinoma cell line CNE⁃2R after overexpressing miR⁃486⁃5p. Methods microRNA(miRNA) sequencing and RT⁃qPCR were used to detect the expression level of miR⁃486⁃5p in the parent cell line CNE2 and the radioresistant cell line CNE⁃2R. CNE⁃2R cells were infected with miR⁃486⁃5p overexpress⁃ion and negative control lentivirus, which were defined as CNE⁃2R⁃Mimics group and CNE⁃2R⁃NC group, respectively. CCK⁃8 assay and Clonogenic assay were used to detect the proliferation ability and the viability of cells irradiated with different dose of (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy) X⁃ray. The cell apoptosis was detected by Annexin V⁃APC/7⁃AAD double stain assay. Results miRNA sequencing and RT⁃qPCR results showed that the level of miR⁃486⁃5p in cell line CNE⁃2R was significantly lower than that in parent cell line CNE2. CCK⁃8 assay revealed that the cell proliferation of CNE⁃2R cells overexpressing miR⁃486⁃5p in CNE⁃2R⁃Mimics group was decreased compared with that in the CNE⁃2R⁃NC group after 48 h, 72 h and 96 h culture (all P<0.05). After the cells were irradiated with 2 Gy, 4 Gy, 6 Gy and 8 Gy irradiation, the survival fraction of CNE⁃2R⁃Mimics group was lower than that of the CNE⁃2R⁃NC group (all P<0.05) , with the largest difference in the 8 Gy dose (t=30.152, P<0.001). Flow cytometry analysis indicated that the apoptosis rate of the CNE⁃2R⁃Mimics group was higher than that of  CNE⁃2R⁃NC group at the irradiation dose of 0 Gy and 8 Gy (all P<0.05), and the difference of apoptosis rate under the irradiation dose of 8 Gy was more significant (t=14.945, P<0.001). The Clonogenic assay revealed that the clone formation fraction of the CNE⁃2R⁃Mimics group was lower than that of the CNE⁃2R⁃NC group under different doses, in which the radiobiological parameters D0, Dq, and the survival fraction in irradiation of 2 Gy in CNE⁃2R⁃Mimics group were lower than that of the CNE⁃2R⁃NC group (all P<0.05). Conclusions The nasopharyngeal carcinoma radioresistant cell line CNE⁃2R overexpressing miR⁃486⁃5p may increase radiosensitivity by inhibiting cell proliferation and promoting apoptosis.

Key words: Nasopharyngeal carcinoma, miR-486-5p, Radiosensitivity, Proliferation, Apoptosis

中图分类号: 

  • R739.63