中华放射医学与防护杂志
中華放射醫學與防護雜誌
중화방사의학여방호잡지
Chinese Journal of Radiological Medicine and Protection
2015年
3期
177-182
,共6页
王鹏飞%黎文汉%洪顺明%张春智
王鵬飛%黎文漢%洪順明%張春智
왕붕비%려문한%홍순명%장춘지
恶性胶质瘤%放射抗性%MiR-221/222%DNA损伤%Akt
噁性膠質瘤%放射抗性%MiR-221/222%DNA損傷%Akt
악성효질류%방사항성%MiR-221/222%DNA손상%Akt
Glioblastoma%Radiation resistance%MiR-221/222%DNA damage%Akt
目的 探讨miR-221/222增加恶性胶质瘤放射抗性的机制.方法 实时荧光定量PCR(real-time PCR)技术检测U251、U87及LN229系胶质瘤细胞在接受照射后3和6 h miR-221/222的表达水平;通过平板克隆形成实验,观察敲低U251、U87及LN229系胶质瘤细胞miR-221/222后肿瘤细胞放射敏感性的变化;染色质免疫沉淀技术(ChIP)检测c-jun与miR-221/222的结合情况;虫荧光素酶活性报告试验验证PTEN是miR-221/222的靶基因;使用Western blot检测敲低胶质瘤细胞miR-221/222后的相关蛋白的表达;通过胶质瘤裸鼠模型,观察敲低miR-221/222后放射治疗的效果.结果 U251、U87及LN229系胶质瘤细胞在接受照射后miR-221/222的表达上调(t=5.48 ～ 29.21,P＜0.05);敲低胶质瘤细胞miR-221/222后,其放射敏感性提高(F=1 202.22,1 789.12,1 012.32,P＜0.05); c-jun转录调控miR-221/222的表达;PTEN是miR-221/222的靶基因(t=13.16,P＜0.05);Western blot检测显示,敲低胶质瘤细胞miR-221/222后,pAkt及DNA-PKcs的表达下调,PTEN和GSK-3β的表达上调,Akt表达保持稳定;体内实验结果显示,敲低miR-221/222的表达联合放射治疗可显著抑制胶质瘤的生长(F =56.36,P＜0.05).结论 放射可诱导胶质瘤细胞miR-221/222的表达上调,miR-221/222通过激活Akt通路增加恶性胶质瘤的放射抗性.
目的 探討miR-221/222增加噁性膠質瘤放射抗性的機製.方法 實時熒光定量PCR(real-time PCR)技術檢測U251、U87及LN229繫膠質瘤細胞在接受照射後3和6 h miR-221/222的錶達水平;通過平闆剋隆形成實驗,觀察敲低U251、U87及LN229繫膠質瘤細胞miR-221/222後腫瘤細胞放射敏感性的變化;染色質免疫沉澱技術(ChIP)檢測c-jun與miR-221/222的結閤情況;蟲熒光素酶活性報告試驗驗證PTEN是miR-221/222的靶基因;使用Western blot檢測敲低膠質瘤細胞miR-221/222後的相關蛋白的錶達;通過膠質瘤裸鼠模型,觀察敲低miR-221/222後放射治療的效果.結果 U251、U87及LN229繫膠質瘤細胞在接受照射後miR-221/222的錶達上調(t=5.48 ～ 29.21,P＜0.05);敲低膠質瘤細胞miR-221/222後,其放射敏感性提高(F=1 202.22,1 789.12,1 012.32,P＜0.05); c-jun轉錄調控miR-221/222的錶達;PTEN是miR-221/222的靶基因(t=13.16,P＜0.05);Western blot檢測顯示,敲低膠質瘤細胞miR-221/222後,pAkt及DNA-PKcs的錶達下調,PTEN和GSK-3β的錶達上調,Akt錶達保持穩定;體內實驗結果顯示,敲低miR-221/222的錶達聯閤放射治療可顯著抑製膠質瘤的生長(F =56.36,P＜0.05).結論 放射可誘導膠質瘤細胞miR-221/222的錶達上調,miR-221/222通過激活Akt通路增加噁性膠質瘤的放射抗性.
목적 탐토miR-221/222증가악성효질류방사항성적궤제.방법 실시형광정량PCR(real-time PCR)기술검측U251、U87급LN229계효질류세포재접수조사후3화6 h miR-221/222적표체수평;통과평판극륭형성실험,관찰고저U251、U87급LN229계효질류세포miR-221/222후종류세포방사민감성적변화;염색질면역침정기술(ChIP)검측c-jun여miR-221/222적결합정황;충형광소매활성보고시험험증PTEN시miR-221/222적파기인;사용Western blot검측고저효질류세포miR-221/222후적상관단백적표체;통과효질류라서모형,관찰고저miR-221/222후방사치료적효과.결과 U251、U87급LN229계효질류세포재접수조사후miR-221/222적표체상조(t=5.48 ～ 29.21,P＜0.05);고저효질류세포miR-221/222후,기방사민감성제고(F=1 202.22,1 789.12,1 012.32,P＜0.05); c-jun전록조공miR-221/222적표체;PTEN시miR-221/222적파기인(t=13.16,P＜0.05);Western blot검측현시,고저효질류세포miR-221/222후,pAkt급DNA-PKcs적표체하조,PTEN화GSK-3β적표체상조,Akt표체보지은정;체내실험결과현시,고저miR-221/222적표체연합방사치료가현저억제효질류적생장(F =56.36,P＜0.05).결론 방사가유도효질류세포miR-221/222적표체상조,miR-221/222통과격활Akt통로증가악성효질류적방사항성.
Objective To study the pathway of miR-221/222 in enhancing radiation resistance of glioblastoma.Methods After 2 Gy of X-ray irradiation,the expressions of miR-221/222 in U251,U87 and LN229 glioblastma cells were detected with real-time PCR.Clonogenic assay was used to measure the radiosensitivity of glioblastoma after knocking down miR-221/222.ChIP assay was used to identify the combination situation of c-jun and miR-221/222.Luciferase assay was applied to check whether PTEN was a target of miR-221/222.Western blot was used to detect the expression of relevant proteins in the glioblastoma cells after knocking down miR-221/222.The effect of miR-221/222 and irradiation on growth of glioblastoma in nude mice was also observed.Results The expression of miR-221/222 was increased by irradiation(t =5.48 ～29.21,P ＜ 0.05) and the radiosensitivity of anti-miR-221/222-transfected cells was alsoincreased(F=1 202.22,1 789.12,1 012.32,P＜0.05).MiR-221/ 222 was transcriptionally regulated by c-jun with a target of PTEN (t =13.16,P ＜ 0.05).When miR-221/222 was knocked-down,the expression of pAkt and DNA-PKcs were down-regulated while PTEN and GSK-3β were up-regulated,and the expression of Akt were not changed.Moreover,the growth of xenograft tumor was significantly inhibited by the combination treatment of anti-miR-221/222 and irradiation(F =56.36,P ＜ 0.05).Conclusions The expression of miR-221/222 in glioblastoma cells can be increased by irradiation,and the activation of Akt pathway downstream miR-221/222 could enhance the radiation resistance of glioblastoma.
