Aurora-A/NF-κB参与人肺腺癌耐药株SPC-A1/多西他赛的放疗抵抗研究

Aurora-A/NF-κB is involved in the radioresistance of human lung adenocarcinoma SPC-A1/DTX cells

目的人肺腺癌SPC-A1的多药耐药株SPC-A1/多西他赛(docetaxel,DTX)与亲本株SPC-A1相比,该耐药株同时具有放疗抵抗。文中探讨Aurora-A/NF-κB在SPC-A1/DTX放疗抵抗中的作用及潜在分子机制。方法以人肺腺癌耐药株SPC-A1/DTX为研究对象,分为sh-Aurora-A组(Aurora-A干扰质粒转染)、sh-NC组,NF-κB抑制组(SPC-A1/DTX+NF-κB抑制剂)和对照组(DMSO),分别用MTT法测定以上所有组的体外放疗敏感性,克隆实验检测各组细胞体外增殖能力,实时定量RT-PCR及Western blot法分别检测目的基因的mRNA及蛋白的表达。结果 1SPC-A1和SPC-A1/DTX细胞对放疗的半数致死量(50%effective dose,ED50)值分别为(6.5±0.3)Gy、(12.8±0.6)Gy,两者差异有统计学意义(P<0.01);在0、2、4、6 Gy放疗条件下,SPC-A1细胞克隆数分别为(345±20)个、(252±22)个、(170±15)个和(81±10)个,SPC-A1/DTX细胞克隆数分别为(402±21)个、(370±18)个、(301±16)个、(252±15)个,同放疗条件下SPC-A1细胞较SPC-A1/DTX细胞体外增值能力明显下降(P<0.05)。2SPC-A1/DTX细胞中的Aurora-A蛋白(1.00±0.08)、mRNA(1.00±0.06)水平较SPC-A1细胞(0.49±0.03、0.22±0.02)均明显升高(P<0.05)。3MTT法测定sh-NC组和sh-Aurora-A组细胞的ED50值分别为(11.8±0.5)Gy、(7.1±0.3)Gy,差异具有统计学意义(P<0.01);4MTT法测定对照组和NF-κB抑制组细胞的ED50值分别为(11.7±0.5)Gy、(6.1±0.3)Gy,差异有统计学意义(P<0.01)。5抑制Aurora-A后,发现IκBa为原表达的(2.18±0.32)倍,NF-κB为原表达的(0.24±0.03)倍;与亲本株SPC-A1的IκBa(1.00±0.05)和NF-κB(1.00±0.04)比较,耐药株SPC-A1/DTX中IκBa低表达(0.65±0.04),NF-κB呈高表达(2.18±0.15),差异均有统计学意义(P<0.01)。结论 Aurora-A/NF-κB参与了人肺腺癌耐药株SPC-A1/DTX的放疗抵抗。

Objective Human lung adenocarcinoma SPC-A1 /DTX cells have a higher radioresistance than SPC-A1 cells.This study was to investigate the role of Aurora-A / nuclear factor κB（ NF-κB） in the radioresistance of human lung adenocarcinoma SPC-A1 / DTX cells and its possible molecular mechanisms. Methods We collected human lung adenocarcinoma SPC-A1 and SPC A1 / DTX cells and divided them into four groups： sh-Aurora-A（ Aurora-A plasmid interference）,sh-NC,NF-κB inhibition（ SPC-A1 /DTX ＋ NF-κB inhibitor）,and DMSO control. We measured the in vitro radio-sensitivity of the cells by MTT assay,determined their proliferation ability by cloning assay,and detected the mRNA and protein expressions of the target genes by real-time quantitative RTPCR and Western blot,respectively. Results The 50% effective doses（ ED50） of the SPC-A1 and SPC-A1 /DTX cells on radiotherapy were（ 6. 5 ± 0. 3） and（ 12. 8 ± 0. 6） Gy,respectively,with statistically significant difference between the two groups（ P〈0. 01）. In the radiation doses of 0,2,4,and 6 Gy,the numbers of the cloned SPC-A1 cells were 345 ± 20,252 ± 22,170 ± 15,and 81 ± 10,significantly lower than those of the cloned SPC-A1 / DTX cells（ 402 ± 21,370 ± 18,301 ± 16,and 252 ± 15）（ P〈0. 05）. The protein and mRNA expressions of Aurora-A were remarkably higher in the SPC-A1 / DTX than in the SPC-A1 cells（ 1. 00 ± 0. 08 and 1. 00 ± 0.06 vs 0. 49 ± 0. 03 and 0. 22 ± 0. 02,P〈0. 05）. MTT assay showed a higher ED50 in the sh-NC than in the sh-Aurora-A cells（ [11.8 ± 0. 5] vs [7. 1 ± 0. 3] Gy,P〈0. 01） as well as in the control than in the NF-κB inhibition group（ [11. 7 ± 0. 5] vs [6. 1 ± 0. 3]Gy,P〈0. 01）. Inhibition of Aurora-A increased the expression of IκBa by 2. 18 ± 0. 32 times（ P〈0. 01） and that of NF-κB by 0. 24± 0. 03 times（ P〈0. 01）. The expressions of IκBa（ 1. 00 ± 0. 05） and NF-κB（ 1. 00 ± 0. 04） were significantly lower in the parent strains of SPC-A1 than 0. 65 ± 0. 04 and 2. 18 ± 0. 15 in the drug-resistant strains of SPC-A1 / DTX（ P〈0. 01）. Conclusion Aurora-A / NF-κB is involved in the radioresistance of human lung adenocarcinoma SPC-A1 / DTX cells.