EGFR和NRF2对电离辐射导致的DNA损伤修复的影响

The effects of EGFR and NRF2 on DNA damage repair induced by ionizing radiation

    摘要
  • 摘要:
    目的 探讨表皮生长因子受体（EGFR）和核转录因子E2相关因子2（NRF2）对人宫颈癌HeLa细胞受到电离辐射损伤后的DNA损伤响应及修复作用。
    方法 将人宫颈癌HeLa细胞按2种处理方式分组：（1）采用小干扰RNA敲降HeLa细胞中的EGFR，采用137Cs γ射线照射源照射细胞。将HeLa细胞分为对照组（HeLa siCtrl）、敲降EGFR组（HeLa siEGFR）、照射组（HeLa siCtrl+8 Gy）、敲降EGFR+照射组（HeLa siEGFR+8 Gy）。采用免疫荧光实验（8 Gy照射后6、12、24 h）检测细胞中磷酸化组蛋白H2A变异体（γ-H2AX）foci的数量；采用实时荧光定量聚合酶链式反应（RT-qPCR）检测NRF2下游靶基因；采用流式细胞术检测EGFR对HeLa细胞周期的影响；采用核质分离实验分离HeLa细胞的胞质蛋白和胞核蛋白；采用蛋白质免疫印迹法检测NRF2、EGFR、血红素氧合酶1（HO-1）、共济失调毛细血管扩张突变基因Rad3相关激酶（ATR）Thr1989位点的磷酸化水平、检查点激酶1（CHK1）在Ser345位点的磷酸化水平。（2）采用小干扰RNA敲降HeLa细胞中的NRF2，采用137Cs γ射线照射源照射细胞。将HeLa 细胞分为对照组（HeLa siCtrl）、敲降NRF2组（HeLa siNRF2）、照射组（HeLa siCtrl+8 Gy）、敲降NRF2+照射组（HeLa siNRF2+8 Gy）。采用免疫荧光实验检测细胞中γ-H2AX foci的数量。符合正态分布的计量资料的组间比较采用两独立样本t检验（方差齐）。
    结果 （1）8 Gy照射6、12、24 h后，HeLa siEGFR+8 Gy组细胞中γ-H2AX foci的数量均多于HeLa siCtrl组（94.00±1.00）% 对（89.67±2.03）%、（72.33±1.76）% 对（60.00±1.73）%、（43.00±2.31）% 对（26.33±1.20）%，且差异有统计学意义（t=3.919、4.919、6.402，均P<0.05）。与HeLa siCtrl组比较，HeLa siEGFR+8 Gy组的细胞G2/M期阻滞显著受损（46.53±3.06）%对（37.90±4.61）%，且差异有统计学意义（t=4.384，P<0.05）。与HeLa siCtrl组比较，HeLa siEGFR+8 Gy组的HO-1表达下降66.66%（1.35±0.10对0.45±0.02），且差异有统计学意义（t=8.782，P<0.05）。敲降EGFR后细胞核内的NRF2蛋白水平降低，辐射引起的NRF2下游ATR-CHK1信号通路活化水平及HO-1蛋白水平均降低。（2）8 Gy照射6、12、24 h后，与HeLa siCtrl组相比，HeLa siNRF2+8 Gy组细胞中γ-H2AX foci的数量均多于HeLa siCtrl组（96.67±0.88）%对（89.67±2.03）%、（77.33±1.20）% 对（60.00±1.73）%、（54.33±2.19）% 对（26.33±1.20）%，且差异均有统计学意义（t=3.166、4.919、11.220，均P<0.05）。
    结论 电离辐射条件下，敲降EGFR可以减少NRF2蛋白入核，抑制ATR-CHK1信号通路激活及下游基因HO-1的表达，降低人宫颈癌HeLa细胞的DNA损伤修复能力。

     

    Abstract:
    Objective To investigate the effect of epidermal growth factor receptor (EGFR) and nuclear factor-E2-related factor 2(NRF2) on DNA damage response and repair in human cervical cancer HeLa cells after exposure to ionizing radiation.
    Methods Human cervical cancer HeLa cells were treated in two groups. (1) EGFR was knocked down in HeLa cells using a small interfering RNA, and the cells were irradiated using a 137Cs γ-ray irradiation source. HeLa cells were divided into control group (HeLa siCtrl), knockdown EGFR group (HeLa siEGFR), irradiation group (HeLa siCtrl+8 Gy), and knockdown EGFR+irradiation group (HeLa siEGFR+8 Gy). The number of phosphorylated histone 2A variant (γ-H2AX) foci in the cells was detected by immunofluorescence assay (6, 12, and 24 h after 8 Gy irradiation); the NRF2 downstream target genes were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) assay; the expression levels of flow cytometry were used to detect the effect of EGFR on HeLa cell cycle; nucleoplasmic separation assay was used to isolate cytoplasmic and cytosolic proteins from HeLa cells; protein immunoblotting was used to detect the phosphorylation levels of NRF2, EGFR, HO-1, ataxia-telangiectasia mutated gene and Rad3-related kinase (ATR) Thr1989 locus, cell cycle checkpoint kinase 1 (p-CHK1), and Ser345 site phosphorylation level. (2) A small interfering RNA was used to knock down NRF2 in HeLa cells, and the cells were irradiated with a 137Cs γ-ray irradiation source. HeLa cells were divided into control group (HeLa siCtrl), knockdown NRF2 group (HeLa siNRF2), irradiation group (HeLa siCtrl+8 Gy), and knockdown NRF2+ irradiation group (HeLa siNRF2+8 Gy). An immunofluorescence assay was used to detect the number of γH2AX foci in HeLa cells. Inter-group comparisons of measures conforming to normal distribution were performed by two independent sample t-tests (chi-squared).
    Results (1) After 8 Gy irradiation for 6, 12, and 24 h, the number of γH2AX foci in HeLa siEGFR+8 Gy were more than that HeLa siCtrl ((94.00±1.00)% vs. (89.67±2.03)%, (72.33±1.76)% vs. (60.00±1.73)%, (43.00±2.31)% vs. (26.33±1.20)%), and the differences were statistically significant (t=3.919, 4.989, 6.402; all P<0.05). The HeLa siEGFR+8 Gy impaired radiation-induced G2/M phase cell cycle block compared with the HeLa siCtrl, ((46.53±3.06)% vs. (37.90±4.61)%), and the difference was statistically significant (t=4.384, P<0.05). Compared with the HeLa siCtrl, HeLa siEGFR+8 Gy inhibited the radiation induced decrease in HO-1 expression by 66.66%(1.35±0.10 vs. 0.45±0.02), and the difference was statistically significant (t=8.782, P<0.05). The level of NRF2 protein in the nucleus was reduced after knocking down EGFR, and the radiation-induced activation level of the ATR-CHK1 signaling pathway downstream of NRF2 and the level of HO-1 protein were reduced. (2) After 8 Gy irradiation for 6, 12, and 24 h, the number of γH2AX foci in HeLa siNRF2+8 Gy was more than that HeLa siCtrl ((96.67±0.88)% vs. (89.67±2.03)%, (77.33±1.20)% vs. (60.00±1.73)%, (54.33±2.19)% vs. (26.33±1.20)%), and all differences were statistically significant (t=3.166, 4.989, 11.220; all P<0.05).
    Conclusions Under ionizing radiation conditions, knocking down EGFR can reduce the nuclear translocation of NRF2 protein, inhibit the activation of the ATR-CHK1 signaling pathway and the downstream expression of the HO-1 gene, and decrease the DNA damage repair capacity of human cervical cancer HeLa cells.

     

    • HTML全文
    • 参考文献(17)
    • 相关文章
    • 施引文献
  • 资源附件(0)

/

返回文章
返回