微小 RNA -150对鼻咽癌细胞放疗抵抗的影响研究

摘要

Objective To investigate the effect of miR-150 on the radioresistance of nasopharyngeal carcinoma (NPC)cells and analyze its mechanism. Methods From January 2014 to June 2015,we established a radioresistant NPC cell line CNE - 2R. CNE - 2 and CNE - 2R were taken,and clone formation assay was conducted to calculate cloning formation efficiency(PE)and cell survival rate with different radiotherapy doses(0,3 and 6 Gy). MTS method was employed to detect cell proliferation activity on day 1,day 2,day 3,day 4 and day 5. Real - time PCR method was used to detect the levels of miR-150 and GSK3β mRNA,and GSK3β level was measured by Western blotting method. CNE - 2 cells were randomly divided into miR-150 mimics group( transfection of miR-150 mimics) and miRs irrelevant sequence group( transfection of miRs irrelevant sequence),and luciferase activity was detected by luciferasereport carrier experiment. Results CNE - 2 and CNE -2R were not significantly different in PE and cell survival rate when the radiation dose was 0 Gy(P > 0. 05). CNE - 2R was higher than CNE - 2 in PE and cell survival rate when the radiation doses were 3 Gy and 6 Gy(P < 0. 05). After radiation for 1 day to 5 days,the activity of CNE - 2R cell proliferation activity was higher than CNE - 2(P < 0. 05). CNE - 2R was higher in miR-150 and lower in GSK3β mRNA than CNE - 2(P < 0. 05). CNE - 2R was lower than CNE - 2 in the level of GSK3β(P< 0. 05). After the transfection of GSK3β wild type plasmid,miR-150 mimics group was lower than miRs irrelevant sequence group in luciferase activity(P < 0. 05);after the transfection of GSK3β mutant plasmid,miRs irrelevant sequence group and miR-150 mimics group were not significantly different in luciferase activity(P > 0. 05). Conclusion miR-150 is involved in the radioresistance of NPC cells. GSK3β is a direct target gene of miR-150,and miR-150 - GSK3β axis may be a possible strategy for rational NPC treatment.%目的：探讨微小 RNA（miR）-150对于鼻咽癌细胞放疗抵抗的影响，并分析其机制。方法2014年1月—2015年6月，建立放疗抵抗的鼻咽癌细胞株 CNE -2R。取 CNE -2和 CNE -2R，通过克隆形成实验计算不同放疗剂量下（0、3、6 Gy）的克隆形成率（PE）、细胞存活率，采用 MTS 法检测照射1、2、3、4、5 d 时的细胞增殖活性，实时定量荧光聚合酶链式反应（ Real - time PCR）法检测miR-150、糖原合成酶激酶3β（ GSK3β） mRNA 水平， Western blotting 法检测 GSK3β水平。将 CNE -2随机分为 miR-150模拟物组（转染 miR-150模拟物）、微小 RNAs （miRs）无关序列组（转染 miRs 无关序列），通过荧光素酶报告载体实验检测荧光素酶活性。结果0 Gy 放疗剂量时， CNE -2与 CNE -2R 的 PE、细胞存活率比较，差异无统计学意义（P >0.05）；3 Gy、6 Gy 放疗剂量时，CNE -2R 的PE、细胞存活率高于 CNE -2（P <0.05）。照射1~5 d 时，CNE -2R 的细胞增殖活性高于 CNE -2（P <0.05）。CNE-2R 中miR-150水平高于 CNE -2，GSK3β mRNA 水平低于 CNE -2（P <0.05）。CNE -2R 中 GSK3β水平低于 CNE -2（P <0.05）。转染 GSK3β野生型质粒后，miR-150模拟物组荧光素酶活性低于 miRs 无关序列组（ P <0.05）；转染GSK3β突变型质粒后，miRs 无关序列组与miR-150模拟物组荧光素酶活性比较，差异无统计学意义（P >0.05）。结论miR-150参与了鼻咽癌细胞的放疗抵抗，GSK3β是miR-150的直接靶基因，miR-150- GSK3β轴可能成为一种新的用于合理治疗鼻咽癌的策略。