泛素连接酶RING2对苯并[a]芘染毒人支气管上皮细胞周期和P53蛋白表达的影响

Role of ubiquitin protein ligase RING2 in cell cycle and expressions of P53 in human bronchial epithelia cells exposed to benzo[a]pyrene

目的:探讨泛素连接酶RING2对苯并[a]芘(Ba P)染毒人支气管上皮16HBE细胞周期和P53蛋白表达的影响。方法:以16HBE未处理组为阴性对照组,二甲基亚砜(DMSO)组为溶剂对照组,MOCK组为序列对照组。在使用RNA干扰技术降低16HBE细胞泛素连接酶RING2基因表达前后,分别采用不同浓度Ba P(1、2、4、8、16、32μmol/L)染毒24 h;或16μmol/L Ba P染毒不同时间(1、2、4、8、12、24 h)。用流式细胞术检测干扰前后两组细胞周期分布情况,用Western-blot法检测干扰前后两组细胞P53蛋白表达水平。结果:流式细胞术检测结果显示,与阴性对照组比较,16HBE细胞染毒后各浓度和各时点组S期细胞所占的比例均增加(P<0.05),而16HBE(si RNA-RING2)各浓度和各时点组S期细胞所占的比例均下降(P<0.05)。协方差分析显示分组因素(是否进行RNAi)和染毒浓度都对S期细胞比例有影响(P均<0.01),16HBE(si RNA-RING2)细胞组的修正均数(17.09%)比16HBE细胞组(31.55%)明显降低(P<0.01)。分组因素和染毒时间都对S期细胞比例有影响(P均<0.01),16HBE(si RNA-RING2)细胞组的修正均数(13.07%)比16HBE细胞组(28.04%)明显下降(P<0.01)。Western-blot结果显示,与阴性对照组比较,16HBE细胞染毒后各浓度和各时点组P53的表达水平均增加(P<0.05),而16HBE(si RNA-RING2)细胞除16μmol/L染毒8 h组外,其余各组P53的表达水平均降低(P<0.05)。协方差分析显示分组因素和染毒浓度都对P53的表达水平有影响,P值分别为0.026和0.028。16HBE(si RNA-RING2)细胞组的修正均数(0.989)比16HBE细胞组(1.375)明显下降(P<0.05);分组因素和染毒时间都对P53的表达水平有影响,P值分别为0.007和0.035。16HBE(si RNA-RING2)细胞组的修正均数(0.857)比16HBE细胞组(1.541)明显下降(P<0.05)。结论:RING2参与的组蛋白泛素化修饰可能通过影响P53表达和细胞周期S期的变化来发挥对DNA损伤修复的调控。

OBJECTIVE： To investigate the role of ubiquitin protein ligase RING2 in cell cycle and expression of P53 in human bronchial epithelial cells exposed to benzo[a]pyrene. METHODS： The untreated 16HBE cellsgroups were used as negative control groups, the DMSO groups were used assolvent control groups, the MOCK groups were used as sequence control groups. 16HBE cells and 16HBE（siRNA-RING2） cells were exposed to BaP at different concentrations （1, 2, 4, 8, 16, 32 μmol/L） for 24 h, and were exposed to BaP at 16 p.mol/L for different time points （1, 2, 4, 8, 12, 24 h）. Flow cytometry was used to evaluate the cell cycle. The levels of P53 protein were detected by Western- blot. RESULTS： After BaP treatment, compared with the negative control groups, at each concentration and each time point the proportion of S phase of 16 HBE cells were significantly increased （P〈0.01）. However at each concentration and each time point 16HBE （siRNA-RING2） cells groups the proportion of S phase cells were significantly decreased （P〈0.01）. Covariance analysis shows grouping factors （with or without RNAi） and all concentrations had impact on the proportion of S phase, P values were both less than 0.01. The estimated mean of the proportion of S phase in 16HBE （siRNA-RING2） cellgroup （17.09%） was significantly lower than that of 16HBE cell group （31.55%）. Grouping factors （with or without RNAi） and the exposure time all have impacted on theproportion of S phase, P values were both less than 0.01. The estimated mean of the proportion of S phase in 16HBE （siRNA-RING2） cell group （13.07%） was significantly lower than that of 16HBE cell group （28.04%） （P〈0.05）. After BaP treatment, compared with the negative control group, at each concentration and each time point P53 protein levels at 16 HBE cell groups were significantly increased （P〈0.05）. However, at each concentration and each time point 16HBE （siRNA-RING2） cell groups （except 16 ttmol/L BaP treated 8 h） the levels of P53 protein were significantly decreased （P〈0.05）. Covariance analysis shows grouping factors （with or without RNAi） and all concentration showed impact on the level of P53 protein, P values were 0.026 and 0.028, respectively. The estimate mean of the levels of P53 protein in 16HBE （siRNA-RING2） cell group （0.989） was significantly lower than that of 16HBE cell group （1.375） （P〈0.05）. Grouping factors （with or without RNAi） and the exposure time both impacted on the level of P53 protein, P values were 0.007 and 0.035, respectively. The estimate mean of the levels of P53 protein in 16HBE （siRNA-RING2） cell group （0.857） was significantly lower than that of 16HBE cell group （1.541） （P〈0.05）. CONCLUSION： The histone ubiquitination modifications in which RING2 was involved may regulate DNA repair by affecting the expression of P53 and cell cycle S phase changes.