The Role Of H19/SAHH/DNMT1 Axis In DNA Damage Of BEAS-2B Cells Exposed To BaP

Objective:To study the role of lncRNA H19,SAHH and DNMT1 to regulate OGGI methylation,DNA oxidative damage and cell cycle arrest in BEAS-2B cells treated with BaP,Method:Six low expression(si-H19,si-SAHH,si-DNMT1,si-H19+si-SAHH,si-H1P+si-DNMT1,si-SAHH+si-DNMTI)cell models and a control group wers constructed by siRNA.Western blot was used to detect the levels of DNMTs.ELISA was used to detect DNMT1 activity and the contents of 8-OHdG.The interaction between SAHH and DNMT1 was detected by Co-IP.The cellular co-localization between H19,SAHH and DNMT1 was observed by FISH combined with IF.The methylation levels of OGGI gene were detected by using pyrosequencing.The cell cycle was detected by using flow cytometry.Result:After treated with BaP in BEAS-2B cells,the expressions of DNMT1 were increased at a dose-and time-dependent manner(P<0.05),which was 58%higher at 32 ?mol/L BaP for 24h compared with the solvent control.The expressions of DNMT3B were decreased(P<0.05),which were reduced by 39.4%at 32 ?mol/L BaP for 24h compared with the solvent control.However,the expression of DNMT3A did not change significantly(P>0.05).After exposed to BaP,compared with WT cells,the expressions and activity of DNMT1 in all kinds of low-expressing(si-H19,si-SAHH,si-H19+si-SAHH)cells were significantly reduced(P<0.05).There were interactions between transfection and BaP exposure(P<0.05).As analysis of covariance to control exposure concentration factors showed that the estimate means of DNMT1 protein expressions(0.68)and activity(0.62)in si-H19+si-SAHH cells were significantly lower than those of WT cells(1.18 and 1.18,respectively)(P<0.05).Co-IP analysis indicated that after BaP exposure,compared with WT cells,the contents of SAHH in DNMT1-containing proteins were significantly increased by 42.6%and 40.5%in WT and si-NC cells,respectively(P<0.05).After exposed to BaP,the contents of SAHH in DNMT1-containing proteins in si-H19 cells were increased significantly by 56%(P<0.05)compare to WT cells.There were interactions between between transfection and BaP exposure(P<0.05),As analysis of covariance to control exposure concentration factors showed that the estimate means of the contents of SAHH in DNMT1-containing proteins(1,38)in si-H19 cells was significantly higher than those of WT cells(1.21)(P<0.05).IF results showed that endogenous H19 SAHH and DNMT1 proteins were co-localized mainly in the perinuclear cytoplasm and nucleus after exposed to BaP.We found that the fluorescence intensity of DNMT1 was increased after BaP exposure for 24 hours.What's more,the increased fluorescence intensity of H19 and DNMT1,and SAHH and DNMT1 was found after BaP exposure for 24 hours.In addition,we found the co-localized fluorescence intensities of SAHH and DNMT1 in si-H19 cells exposed to BaP were increased compared with untreated si-H19 cells.Pyrosequencing results indicated that after BaP exposure,compared with WT cells,a significant rise of the levels of OGGI nethylation in WT,si-NC,si-DNMT1,si-H19+si-DNMT1,and si-SAHH + si-DNMT1 cells was observed(P<0.05).At the time,a significant rise of the levels of OGG1 methylation in si-DNMT1 and si-SAHH+si-DNMTI cells whether or not exposed to BaP was also observed(P<0.05).After exposed to BaP,compared with WT cells,the levels of OGGI methylation in si-SAHH+si-DNMT1 cells were significantly elevated(P<0.05).There were interactions between transfection and BaP exposure(P<0.05).As analysis of covariance to control exposure concentration factors showed that the estimate means of OGGI methylation in si-DNMT1 cells(1.28)and si-SAHH+si-DNMT1 cells(1.37)were significantly higher than WT cells(1.14).ELISA results indicated that after BaP exposure,compared with WT cells,the contents of 8-OHdG in WT,si-NC,si-DNMTJ,si-H19+ si-DNMT1,and si-SAHH+si-DNMT1 cells were significantly increased(P<0.05).Compared with the contents of 8-OHdG in si-SAHH+si-DNMT1 cells were significantly increased by 19.2%(P<0.05),while H19/DNMT1 double knockdown(1,15)restored the contents of 8-OHdG(P>0.05).There were interactions between transfection and BaP exposure(P<0.05).As analysis of covariance to control exposure concentration factors showed that the estimate means of the contents of 8-OHdG in si-DNMT1 cells(1.20)and si-SAHH+si-DNMTI cells(1.24)were significantly higher than WT cells(1.15)(P<0.05).FCM results indicated that after BaP exposure,compared with WT cells,cellular proportion in S phase in WT(33.6%),si-NC(34.0%),si-DNMT1(34.6%),si-H19+si-DNMT1(38.0%),and si-SAHH+si-DNMT1(60.2%)cells were increased after exposed to BaP(P<0.05),among which a significant rise of cellular proportion in S phase in si-H19+si-DNMT1 and si-SAHH-si-DNMT1 cells whether or not exposed to BaP(P<0.05).Compared with WT cells exposed to BaP,the cellular proportion in S phase in si-SAHH+si-DNMT1 cells were significantly increased by 22.69%(P<0.05),while si-H19+si-DNMTl cells abrogated this effect(P>0.05).There were interactions between transfection and BaP exposure(P<0.05).As analysis of covariance to control exposure concentration factors showed that the estimate means of the cellular proportion in S phase in si-SAHH+si-DNMT1 cells(1.37)were significantly higher than WT cells(1.24).Conclusion:1.H19 binding to SAHH interacted with DNMT1 and exaggerate its expression and activity in BaP-treated cells.2.The interaction of SAHH and DNMT1 would be strengthened by BaP and inhibition of H19 enhanced the interaction of SAHH and DNMT1 induced by BaP.3.H19/SAHH/DNMT1 plays an important role in the OGG1 methylation,oxidative DNA damage and cell cycle.