RING2对苯并[a]芘导致的人支气管上皮细胞BPDE-DNA加合物及CHK1的影响

Effects of ubiquitin protein ligase RING2 on BPDE-DNA adducts and CHK1 in BEAS-2B cells exposed to benzo(a)pyrene

[目的]探讨泛素连接酶RING2对苯并[a]芘(Ba P)导致的人支气管上皮细胞(BEAS-2B)BPDE-DNA加合物及周期检测点激酶1(CHK1)表达水平的影响。[方法]用不同浓度的Ba P(0、1、2、4、8、16、32μmol/L)染毒BEAS-2B细胞24 h,以16μmol/L Ba P染毒BEAS-2B细胞不同时间(0、1、2、4、8、12、24 h);通过siR NA干扰技术降低BEAS-2B细胞中泛素连接酶RING2的表达水平,用16μmol/L Ba P染毒BEAS-2B和BEAS-2B(siRNA-RING2)细胞24 h。使用免疫组化荧光法检测细胞中BPDE-DNA加合物的表达情况,采用Western blot检测细胞中CHK1及其S345位点磷酸化的水平。[结果]免疫组化荧光法结果显示,与正常对照组相比,转染前后染毒组的荧光强度均增加(P<0.01),提示染毒后BPDEDNA加合物增加,其中BESA-2B(siRNA-RING2)染毒组的荧光强度较正常染毒组的增加了32%。Western blot结果显示,随着染毒时间和染毒浓度的增加,BESA-2B的CHK1及CHK1 S345-p的水平逐渐增加(P<0.01);16μmol/L Ba P染毒BEAS-2B(siRNA-RING2)细胞24 h后,其CHK1及CHK1 S345-p水平较对照组明显下降(P<0.01);析因分析表明,转染与染毒均对CHK1及CHK1 S345-p的表达量有影响,两因素间存在交互作用(P<0.01),转染后染毒组与不染毒组CHK1及CHK1 S345-p水平的差异,与正常细胞组的相比,分别降低了54%和51%;协方差分析控制染毒因素后,BESA-2B(siRNA-RING2)组CHK1及CHK1 S345-p水平的修正均数(分别为0.77和0.89)较BEAS-2B组的修正均数(分别为1.24和1.32)明显降低(P<0.01)。[结论]RING2低表达的细胞DNA对Ba P更加敏感,这提示RING2可能通过影响CHK1及其磷酸化水平来调控细胞周期的进程,进而影响DNA的损伤修复。

[ Objective ] To explore the role of ubiquitin protein ligase RING2 in the changes of BPDE-DNA adducts and checkpoint kinase 1 （CHK1） in human bronchial epithelial cells （BEAS-2B） exposed to benzo（a）pyrene （BaP）.[ Methods ] BESA-2B cells were exposed to BaP at concentrations of 0, 1, 2, 4, 8, 16, and 32μmol/L for 24h, respectively; BESA-2B cells were exposed to 16 μmol/L BaP for 0, 1, 2, 4, 8, 12, and 24h, respectively. The expressions of RING2 were inhibited by small interfering RNA （siRNA） in BEAS-2B cells, then the BESA-2B cells and BESA-2B （siRNA-RING2） cells were exposed to 16 μmol/L BaP for 24 h. BPDE-DNA adducts were evaluated by fluorescent immunohistochemistry. The levels of CHK1 and CHK1 S345-p were detected by Western blot.[ Results ] Compared with the normal control group, the fluorescence intensities were significantly elevated both in the BESA-2B cells and the BESA-2B （siRNA-RING2） cells exposed to BaP （P〈0.01）, indicating elevated levels of BPDE-DNA adducts, and the fluorescence intensity of BESA-2B （siRNA-RING2） cells were increased by 32% compared with the BESA-2B cells. According to results of Western blot, the relative expression levels of CHK1 and CHK1 S345-p in BESA-2B cells were significantly elevated along with increasing concentration and exposure time （P〈0.01）; the levels of CHK1 and CHK1 S345-p in BESA-2B （siRNA-RING2） cells exposed to 16 μmol/L BaP for 24 h were significantly decreased compared with the normal control group （P〈 0.01）. The results of factorial analysis showed both transfection and BaP treatment had impacts on the expression levels of CHK1 and CHK1 S345-p, as well as interactions between these two factors （P〈0.01）. Compared with the BESA-2B cells, the difference between levels of CHK1 and CHK1 S345-p in BESA-2B （siRNA-RING2） cells exposed or non-exposed to BaP decreased by 54% or 51%. The results of covariance analysis showed that the estimated means of the levels of CHK1 and CHK1 S345-p （0.77 and 0.89 respectively） in BESA-2B （siRNA-RING2） cells were significantly lower than those in BESA-2B cells （1.24 and 1.32 respectively） （P〈0.01） with control of BaP exposure.[ Conclusion ] The DNA of siRNA-RING2 cells are much more sensitive to BaP exposure than that of normal cells, which indicates that RING2 may be involved in DNA repair by affecting the expression of CHK1 and CHKI S345-p.