【摘要】 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,GSK3βmRNA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。更多还原

【Abstract】 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 1day 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.更多还原