摘要
目的 探讨用siRNA敲降乙肝病毒X蛋白结合蛋白(HBXIP)的表达对宫颈癌ME-180细胞的增殖能力及放射敏感性的影响。方法 根据不同的处理方法,分别按两种分组方式进行分组。(1)把宫颈癌ME-180细胞分为4组:空白对照组、4 Gy γ射线照射组、HBXIP-siRNA转染组以及HBXIP-siRNA+γ射线照射联合组。采用MTT和克隆形成实验法来检测细胞增殖;采用qRTPCR检测凋亡相关蛋白Bcl-2及Bid的表达;Western blot检测蛋白激酶AKT的磷酸化水平。(2)把宫颈癌ME-180细胞分为3组:空白对照组、HBXIP-siRNA单独处理组、HBXIP-siRNA和AKT共转染组。对3组细胞进行不同剂量的γ射线照射,并采用克隆形成实验方法检测细胞生长。采用Student t-test对数据进行统计学分析,P〈0.05表示差异有统计学意义。结果 MTT实验和克隆形成实验结果显示,与γ射线照射组相比,HBXIP-siRNA转染+γ射线照射联合组的宫颈癌ME-180细胞增殖率明显降低(t=11.63、12.17,均P〈0.01),并伴随抑凋亡蛋白Bcl-2表达的降低(t=10.88,P〈0.01)和促凋亡蛋白Bid表达的增高(t=9.31,P〈0.01)。γ射线照射明显上调了HBXIP蛋白的表达水平和AKT蛋白的磷酸化水平,而转染HBXIP-siRNA则抑制了γ射线照射导致的AKT磷酸化水平的升高。另外,与HBXIP-siRNA单独处理组相比,HBXIP-siRNA和AKT共转染组中HBXIP-siRNA对宫颈癌ME-180细胞增殖的影响显著降低(t=8.96,P〈0.01)。结论 降低HBXIP蛋白表达可以抑制辐照诱导的AKT磷酸化水平,进而降低宫颈癌ME-180细胞的增殖能力,同时增强其放射敏感性。
Objective To explore the effects of (hepatitis B X-interacting protein) HBXIP downregulation on the proliferation and radiosensitivity of cervical cancer ME-180 cells. Method According to the different treatment methods, cervical cancer ME -180 cells were divided into different groups:(1)The cervical cancer ME -180 cells were divided into 4 groups:control group, 4 Gy γ ray irradiation group, HBXIP-siRNA transfection group and HBXIP-siRNA transfection+γ ray irradiation group. cervical cancer ME-180 cell proliferation was detected by MTT and clonogenic assays. The expression of Bcl-2 and Bid mRNA was detected by quantitative real-time polymerase chain reaction, and the phosphorylation of AKT protein was measured by Western blot analysis. (2)The cervical cancer ME-180 cells were divided into 3 groups:control group, HBXIP-siRNA transfection group and HBXIP-siRNA+AKT transfection group. Then the three groups of cells were irradiated with different doses of γ ray, and the cervical cancer ME-180 cell proliferation was detected by clonogenic assay. Statistical significance of the results was determined by SPSS statistical software and analyzed by Student t-test. P〈0.05 were considered statistically significant. Results MTT and clonogenic assays showed that, compared with the cells irradiated alone, the cervical cancer ME-180 cells irradiated in the presence of HBXIP-siRNA had significantly decreased proliferation (t=11.63, 12.17, P〈0.01). The decreased proliferation was accompanied by a decreased expression of Bcl-2 protein (t=10.88, P〈0.01) and an increased expression of Bid protein (t=9.31, P〈0.01). The transfection with HBXIP-siRNA inhibited the increased HBXIP protein expression and AKT phosphorylation, which were caused by radiation. The enhanced AKT expression significantly reduced the HBXIP -siRNA inhibition of cervical cancer ME-180 cell proliferation after irradiation as compared with that of the HBXIP-siRNA alone (t=8.96, P〈0.01). Conclusion HBXIP down -regulation reduced the proliferation and increased the radiosensitivity of cervical cancer ME-180 cells by mediating AKT activation.
作者
姜勉
董佳丽
李航
樊赛军
Jiang Mian Dong Jiali Li Hang Fan Saijun(Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192, China)
出处
《国际放射医学核医学杂志》
2017年第5期340-346,共7页
International Journal of Radiation Medicine and Nuclear Medicine
基金
国家自然科学基金(81572969)
科技部科研院所开发项目(2014EG150134)
关键词
HBXIP
RNA
小分子干扰
宫颈肿瘤
细胞增殖
辐射耐受性
HBXIP
RNA, small interfering
Uterine cervical neoplasms
Cell proliferation
Radiation tolerance