Ubiquitin Ligase Ring2 In Benzo [a] Pyrene Cause Dna Damage Human Bronchial Epithelial Cells And The Role Of Histone Single Ubiquitin Research

Objective Through reducing the expression of Ring2 gene in in human bronchial epithelia(16HBE) cells by RNA interference，investigate the effect of Ring2 in the process of DNAdamage and expression of mono-ubiquitinated histone H2A in 16HBE cells exposed tobenzo[a]pyrene（B[a]P）.Methods Before and after the reducing the expression of Ring2 gene, the 16HBE cells wereexposed to B[a]P at different concentration（0,1,2,4,8,16,32μmol/L）for 24h;and were exposed toB[a]P at the three concentration（2,8,16μmol/L）for different time（0h，1h,2h,4h,8h,12h,24h）;andwere exposed to B[a]P at the three concentration（2,8,16μmol/L）for 2h,and then recovery fordifferent time（0h,1h,2h,4h,8h,12h,24h）. Comet assay was applied to determine the DNAdamage and the levels of DNA damage were evaluated by the Olive Tail Moments(OTM).Wedetected the levels of protein expressions by Western-blot.ResultsⅠ、The normal expression of Ring2 gene1、The result of single cell gel electrophoresis(SCGE)When the 16HBE cells were exposed to B[a]P at different concentration（0,1,2,4,8,16,32μmol/L）for 24h ,the OTM levels increased with the increasing of concentration,and a significant increment was found as compared with the control group(P＜0.01); When the16HBE cells were exposed to B[a]P at the three concentration（2,8,16μmol/L）for different time（0h，1h,2h,4h,8h,12h,24h）, the OTM levels increased with the elongation of exposure time, anda significant increment was found as compared with thecontrol group(P＜0.01); After exposed to2μmol/L B[a]P for 2 hours and then recovery for different time（0h,1h,2h,4h,8h,12h,24h）, theOTM levels falls down, and a significant decrement was found as compared with the controlgroup after recovery for 2h (P＜0.01); After exposed to 8μmol/L B[a]P for 2 hours and thenrecovery for different time（0h,1h,2h,4h,8h,12h,24h）, the OTM levels falls down after increasingat 1h, and a significant decrement was found as compared with the control group after recoveryfor 2h (P＜0.01); After exposed to 16μmol/L B[a]P for 2 hours and then recovery for differenttime（0h,1h,2h,4h,8h,12h,24h）, the OTM levels increases at 1h(P＜0.01)and then falls down,and a significant decrement was found as compared with the control group (P＜0.01) afterrecovery for 2h.2 The result of Western-blotWhen the 16HBE cells were exposed to B[a]P at different concentration（0,1,2,4,8,16,32μmol/L）for 24h , the levels of mono-ubiquitinated histone H2A proteinexpression increased significantly compared with the control group (P＜0.01); When the 16HBEcells were exposed to B[a]P at the concentration 2μmol/Lfor different time, the levels of mono-ubiquitinated histone H2A protein expression increased significantly compared with thecontrol group after exposing for 2h(P＜0.01); After exposed to 8μmol/L B[a]P for differenttime ,the mono-ubiquitinated histone H2A protein levels increased with the elongation ofexposure time, and a significant increment was found as compared with the control group(P＜0.01); After exposed to 16μmol/L B[a]P for different time ,the mono-ubiquitinated histone H2Aprotein levels increased with the elongation of exposure time, and a significant increment wasfound as compared with the control group after exposing for 4h (P＜0.01); After exposed to2μmol/L B[a]P for 2 hours and then recovery for different time, the levels of mono-ubiquitinatedhistone H2A protein expression increased significantly compared with the control group (P＜0.01),and it reachs the summit at the point of 8h; After exposed to 8μmol/L B[a]P for 2 hours andthen recovery for different time, the levels of mono-ubiquitinated histone H2A proteinexpression increased significantly compared with the control group (P＜0.01), and it reachs thesummit at the point of 8h; After exposed to 16μmol/L B[a]P for 2 hours and then recovery fordifferent time, the levels of mono-ubiquitinated histone H2A protein expression increasedsignificantly compared with the control group (P＜0.01) after recovery for 2h.3 The regression analysisWhen the 16HBE cells were exposed to B[a]P at different concentration for 24h, The regressionanalysis showed that the determination coefficient between OTM and the levels ofmono-ubiquitinated histone H2A protein expression was0.910(P＜0.01) and the regressionequation was Y=5.251-278.807X~2+736.392X~3;When the 16HBE cells were exposed to B[a]P atthe concentration 2μmol/Lfor different time, The regression analysis showed that thedetermination coefficient between OTM and the levels of mono-ubiquitinated histone H2Aprotein expression was0.723 (P<0.05)and the regression equation wasY=2.306-38.713X+209.283X~2-234.211X~3(R~2=0.723 P<0.05); When the 16HBE cells wereexposed to B[a]P at the concentration 8μmol/Lfor different time, The regression analysis showedthat the determination coefficient between OTM and the levels of mono-ubiquitinated histoneH2A protein expression was0.77 (P<0.01)and the regression equation wasY=Exp(0.085+11.476X); When the 16HBE cells were exposed to B[a]P at the concentration16μmol/Lfor different time, The regression analysis showed that the determination coefficientbetween OTM and the levels of mono-ubiquitinated histone H2A protein expression was 0.944(P<0.01)and the regression equation was Y=13.269-169.591X+669.054X~2-625.192X~3; Afterexposed to 2μmol/L B[a]P for 2 hours and then recovery for different time, The regressionanalysis showed that the determination coefficient between OTM and the levels ofmono-ubiquitinated histone H2A protein expression was 0.603 (P<0.05)and the regressionequation was Y=1.574-2.753X~3; After exposed to 8μmol/L B[a]P for 2 hours and then recovery for different time, The regression analysis showed that the determination coefficient betweenOTM and the levels of mono-ubiquitinated histone H2A protein expression was 0.503(P<0.05)and the regression equation was Y=7.353-21.907X+41.582X~3; After exposed to16μmol/L B[a]P for 2 hours and then recovery for different time, The regression analysis showedthat the determination coefficient between OTM and the levels of mono-ubiquitinated histoneH2A protein expression was 0.750 (P<0.05)and the regression equation wasY=6.852-23.601X+58.494X~3.ⅡRNAinterferenceTransfection efficiency and the efficiency of gene silencing：The 16HBE cells were treated with50nM RNA interference sequence for 6h,and then cultured for 24h and30h respectively. Thelevel of Ring2 gene expression decreased, and a significant increment was found as comparedwith the control group .The inhibition efficiency of Ring2 gene was 72%.ⅢThe inhibition expression of Ring21、The result of single cell gel electrophoresis(SCGE)When the 16HBE cells were exposed to B[a]P at different concentration（0,1,2,4,8,16,32μmol/L）for 24h ,the OTM levels increased with the increasing of concentration,and a significant increment was found as compared with the control group(P＜0.01); When the16HBE cells were exposed to B[a]P at the three concentration（2,8,16μmol/L）for different time（0h，1h,2h,4h,8h,12h,24h）, the OTM levels increased with the elongation of exposure time, anda significant increment was found as compared with the control group(P＜0.01); After exposedto 2μmol/L B[a]P for 2 hours and then recovery for different time（0h,1h,2h,4h,8h,12h,24h）, theOTM levels falls down, and a significant decrement was found as compared with the controlgroup after recovery for 2h (P＜0.01); After exposed to 8μmol/L B[a]P for 2 hours and thenrecovery for different time（0h,1h,2h,4h,8h,12h,24h）, the OTM levels falls down , and asignificant decrement was found as compared with the control group (P＜0.01); After exposed to16μmol/L B[a]P for 2 hours and then recovery for different time, the OTM levels falls down, anda significant decrement was found as compared with the control group (P＜0.01) after recoveryfor 2h.2 The result of Western-blotWhen the 16HBE cells were exposed to B[a]P at different concentration（0,1,2,4,8,16,32μmol/L）for 24h , there was no statistically significant between the groups ofthe levels of mono-ubiquitinated histone H2A protein expression; When the 16HBE cells wereexposed to B[a]P at the three concentration (2,8,16μmol/L)for different time, there was nostatistically significant between the groups of the levels of mono-ubiquitinated histone H2Aprotein expression ; After exposed to (2,8,16μmol/L )B[a]P for 2 hours and then recovery for different time, here was no statistically significant between the groups of the levels ofmono-ubiquitinated histone H2A protein expression.3 The regression analysisThe regression analysis showed that there was no statistically significant between OTM and thelevels of mono-ubiquitinated histone H2A protein expression in any group.ⅣThe covariance analysis showed that ,compared with the nomal expression of Ring2,theDNA damage caused by B[a]P was more critical when reducing the expression of Ring2,and thedifference was significant.Conclusion: 1 The dose-effect and time-effect manner of DNA damage in 16HBE exposed toB[a]P were observed.2 A close correlation between the DNA damage and the levels of mono-ubiquitinated histoneH2A protein expression in 16HBE cells when exposing to B[a]P was found.3 Ubiquitinligase enzyme Ring2 played an important role in the process of themonoubiquitinated modifications of hisone H2A induced by DNA danmage.