Role Of Ubiquitin Ligase RING2 In DNA Oxidative Damage And P53-P21 Signal Pathway Of Human Brochial Epithelial Cells Induced By Benzo(a)Pyrene

Objective:To investigate the role of ubiquitin ligase RING2 in DNA oxidative damage and P53-P21 signal pathway of human brochial epithelial cells exposed to benzo(a)pyrene, and preliminary reveal the inherent relationship between histone ubiquitination and DNA damage repair.Methods:The untreated 16 HBE and BEAS-2B cells groups were used as negative control groups, the DMSO groups were used as solvent control groups, the MOCK si RNA groups were used as sequence control groups. Before and after reducing the expression of RING2 gene, these two kinds of cells were exposed to Ba P at different concentrations(1, 2, 4, 8, 16,32 μmol/L) for 24 h, and were exposed to Ba P at 16 μmol/L for different time points(1, 2, 4,8, 12, 24h). ELISA was used to evaluate the levels of 8-OH-d G. The levels of protein were detected by Western-blot.Results:Ⅰ. ELISA results show: 1. Compared with the negative control groups, both 16 HBE and16 HBE(si RNA- RING2) cells groups the levels of 8-OH-d G were significantly increased with increasing Ba P concentration, Covariance analysis shows the estimate mean of 16HBE(si RNA- RING2) cells groups(13.312) were significantly higher than 16 HBE cells groups(7.969)(P<0.05); both 16 HBE and 16 HBE(si RNA- RING2) cells groups the levels of 8-OH-d G were significantly increased with increasing Ba P exposure time.Covariance analysis shows the estimate mean of 16 HBE(si RNA- RING2) cells groups(7.803) were significantly higher than 16 HBE cells groups(4.235)(P<0.05). 2. Compared with the negative control groups, the levels of 8-OH-d G were significantly increased with increasing Ba P concentration, Covariance analysis shows the estimate mean of BEAS-2B(si RNA-RING2) cells groups(11.237) were significantly higher than BEAS-2B cells groups(5.766)(P<0.05); both BEAS-2B and BEAS-2B(si RNA- RING2) cells groups the levels of 8-OH-d G were significantly increased with increasing Ba P exposure time,Covariance analysis shows the estimate mean of BEAS-2B(si RNA-RING2) cells groups(7.615) were significantly higher than BEAS-2B cell groups(3.524)(P<0.05).Ⅱ. Western-blot results show: 1. Compared with the negative control groups, both 16 HBE and BEAS-2B cells groups the the levels of P53, P21, p-P53ser15, p-P53ser20, p-P53ser37 protein were significantly increased after exposed to different concentration and different time Ba P(P< 0.05). 2. The comparison of protein expression between Wild type and si RNA-RING2 cells after exposed to Ba P results show: 16 HBE cells groups the levels of P53, P21, p-P53ser15, p-P53ser20, p-P53ser37 protein were significantly higher than 16HBE(si RNA- RING2) cells groups(P<0.05), respectively were 1.811 times, 3.287 times,4.172 times, 3.018 times and 2.118 times. BEAS-2B cell groups the levels of P53, P21,p-P53Ser15, p-P53Ser20, p-P53Ser37 protein were significantly higher than BEAS-2B(si RNA-RING2) cells groups(P<0.05), respectively were 1.978 times, 2.546 times, 7.012 times, 2.774 times and 1.875 times.Conclusion:1. The level of 8-OH-d G increased after human brochial epithelial cells exposed to Ba P.8-OH-d G and Ba P exposure have significant dose effect and time effect. After si RNA-RING2 cells exposed to Ba P, DNA oxidative damage degree aggravating,RING2 may participate in the DNA oxidative damage repair process.2. P53-P21 signal pathway related protein expression increased after human brochial epithelial cells exposed to Ba P. compared with Wild type cells,si RNA-RING2 cells P53-P21 signal pathway related protein expression decrease, the change of p-P53ser15 is especially striking.3. After si RNA-RING2 cells exposed Ba P, DNA oxidative damage degree aggravating may be associated with P53-P21 signal pathway related protein expression decrease.