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  

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

作　　者：宋阳光 孙永楚 覃月兰 陈凯华[1] 赖林 陈锡山 朱小东[1,3] SONG Yangguang;SUN Yongchu;QIN Yuelan;CHEN Kaihua;LAI Lin;CHEN Xishan;ZHU Xiaodong(Department of Radio-therapy,Guangxi Medical University Cancer Hospital,Nanning 530021,China;Department of Oncology,Liuzhou Worker′s Hospital/Guangxi Medical University Fourth Affiliated Hospital,Liuzhou 545005,China;Department of Oncology,Guangxi Medical University affiliated Wuming Hospital,Nanning 530100,China)

机构地区：[1]广西医科大学附属肿瘤医院放疗科,南宁530021 [2]柳州市工人医院/广西医科大学第四附属医院肿瘤科,柳州545005 [3]广西医科大学附属武鸣医院肿瘤科,南宁530100

出　　处：《中国癌症防治杂志》2023年第1期11-17,共7页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：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)。

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。ObjectiveTo investigate the radiosensitivity effect of radioresistant nasopharyngeal carcinoma cell line CNE-2R after overexpressing miR-486-5p.MethodsmicroRNA(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 overexpression 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.ResultsmiRNA 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 with2 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).ConclusionsThe nasopharyngeal carcinoma radioresistant cell line CNE-2R overexpressing miR-486-5p

关 键 词：鼻咽癌 miR⁃486⁃5p 放射增敏 增殖 凋亡 

分 类 号：R739.63[医药卫生—肿瘤]