Effects Of The Methylation Of OGG1 Regulated By H19/SAHH On The Oxidative DNA Damage In Human Lung Derived Cells Exposed To Benzo[a]pyrene

Objective: To investigate the effects of 8-oxoguanine DNA glycosylase 1(OGG1)methylation regulated by interaction between long non-coding RNA(lnc RNA)H19 and S-adenosylhomocysteine hydrolase(SAHH)on the oxidative DNA damage in human pulmonary cells(16HBE,BEAS-2B,VA13)exposed to Benzo[a]pyrene.In order to provide clues for revealing the epigenetic mechanism of Ba P-induced DNA damage.Methods: The expressions of H19 and SAHH were inhibited by small interfering RNA(si RNA)in human lung derived cells to construct cell models with H19 or SAHH low expression and H19/SAHH double low expression,while the SAHH overexpression cell models were transfected by p CMV-GV141-SAHH(expressing Flag-tagged wild type(WT)SAHH)or p CMV-GV141 expressing the indicated Flag-tagged SAHH mutants(M1-SAHH,M2-SAHH,M3-SAHH).The control group with negative sequence or empty plasmid was set up at the same time.Cells were exposed to Ba P at concentrations of 0,1,2,4,8,16,32 ?mol/L for 24 hours,or were exposed to 16 ?mol/L Ba P for 0,1,2,4,8,12 and 24 hours,and the solvent control group was set at the same time.The relative levels of H19 were detected by reverse transcriptionpolymerase chain reaction(RT-PCR).The distribution of H19 and its co-localization with SAHH were detected by lnc RNA fluorescence in situ hybridization(FISH)and Immunofluorescence(IF)combined with laser scanning confocal microscopy.The interaction between H19 and SAHH was detected by RNA Binding Protein Immunoprecipitation(RIP).The expression and activity of SAHH protein were detected by Western Blot and Spectrophotometry(Fluorescence).8-oxoguanine DNA glycosylase 1(OGG1)methylation was detected by Pyrosequencing.Enzyme linked immunosorbent assay(ELISA)was used to detect the conten t of 8-hydroxy-2 '-deoxyguanosine(8-OHd G)in cells to analyze oxidative DNA damage.Cell cycle was analyzed by flow cytometry(FCM).Results: Results showed that compared to the controls,Ba P-treated cells H19 expressions were increased in a dose-and time-dependent manner(P < 0.05),whereas SAHH protein expressions were decreased(P < 0.05).H19 expressions reached a peak at 32 ?mol/L Ba P for 24 hours,which increased(16HBE 78.0%,VA13 78.4%,BEAS-2B 87.2%)compared with the solvent control group,while SAHH protein expressions decreased to the lowest level(16HBE 78.0%,VA13 78.4%,BEAS-2B 87.2%).lnc RNA FISH and IF results showed that both H19 and SAHH were located in cytoplasm and nucleus,predominantly in perinuclear cytoplasm.After exposed to 16 ?mol/L Ba P for 24 hours,more H19 was detected in perinuclear cytoplasm that expressed lower expressions of SAHH as compared to the u ntreated group,and the immunofluorescence intensity co-localized in cytoplasm and nucleus of H19 and SAHH was significantly increased.RIP results showed that a(5.53-8.46)-fold enrichment of H19 in SAHHcontaining ribonucleoproteins(RNPs)as compared to the untreated group(P < 0.01),whereas GAPDH m RNA did not detectably associate with the SAHH-containing RNPs(P > 0.05).Without Ba P exposure,H19 in SAHH-containing RNPs of WTSAHH,M1-SAHH and M2-SAHH cells were significantly higher than that of WT cells(P < 0.01),and all were significantly increased after 16?mol/L Ba P exposure for 24 hours(P < 0.05).However,H19 in SAHH-containing RNPs of M3-SAHH cells were similar to WT cells treated without or with Ba P exposure(P > 0.05).Compared with untreated group,H19 expressions were increased in all types of SAHH overexpression cells expored to Ba P(P < 0.05),while Ba P-treated M3-SAHH cells did not increased significantly(P > 0.05).With Ba P exposure,M3-SAHH cells H19 expressions were obviously reduced compared to WT-SAHH cells(P < 0.05).When WT and si-NC cells were treated with Ba P to attenuate SAHH protein expressions and activity(P < 0.05),a significant rise in si-H19 cells was observed(expressions 18.5%,activity 94.9%).Factorial analysis shows there are interactions between transfection and Ba P exposure(P < 0.05).As analysis of covariance to control exposure concentration factors show,the estimate means of SAHH protein expressions(1.11)and activity(1.88)in si-H19 cells was significantly higher than those of WT cells(0.85 and 0.87,respectively)(P < 0.05).Pyrosequencing results revealed that a significant increase occurred in OGG1 methylation of WT,si-NC,si-SAHH and si-H19 + si-SAHH cells with Ba P treatment(P < 0.05),whereas a fall was observed when H19 was downregulated(P < 0.05),which decreased(12.5% without and 28.2% with Ba P exposure,respectively)compared to WT cells without Ba P exposure(P < 0.05).Factorial analysis shows there are interactions between transfection and Ba P exposure(P < 0.05).As analysis of covariance to control exposure concentration factors show,the estimate means of OGG1 methylation of si-H19 cells(1.5%)was lower and si-SAHH cells(2.5%)was higher than WT cells(2.2%)(P < 0.05),while H19/SAHH doubleknockdown restored OGG1 methylation(2.1%)to the control level(P > 0.05).ELISA results revealed that the content of 8-OHd G were elevated in all types cells after Ba P treatment(P < 0.05).Compared with WT cells exposed to Ba P,si-H19 cells were decreased by 7.6% and si-SAHH cells were increased by 20.4%(P < 0.05)both exposed to Ba P,while si-H19 + si-SAHH cells were similar to WT cells(P > 0.05).Factorial analysis shows there are interactions between transfection and Ba P exposure(P < 0.05).As analysis of covariance to control exposure concentration factors show,the estimate means of 8-OHd G content of si-H19 cells(1.09)was lower and si- SAHH cells(1.24)was higher than WT cells(1.13)(P < 0.05),while H19/SAHH doubleknockdown restored OGG1 methylation(1.15)to the control level(P > 0.05).FCM results revealed that compared with untreated group,cellular proportion in S phase were increased both in si-SAHH cells(31.1%)and si-H19 + si-SAHH cells(18.1%)not exposed to Ba P(P < 0.05),and were elevated in all types cells expored to Ba P(P < 0.05).Compared with WT cells exposed to Ba P,si-H19 cells were decreased by 13.6% and si-SAHH cells were increased by 13.5%(P < 0.05)both exposed to Ba P,while si-H19 + si-SAHH cells were similar to WT cells(P > 0.05).Factorial analysis shows there are interactions between transfection and Ba P exposure(P < 0.05).As analysis of covariance to control exposure concentration factors show,the estimate means of cellular proportion in S phase of si-SAHH cells(1.40)and si-H19 + si-SAHH cells(1.31)were higher than WT cells(1.18)(P < 0.05),while siH19 cells(1.17)shows no significance(P > 0.05).Conclusions: 1.Ba P exposure promotes H19 binding to SAHH and inhibiting SAHH expression and activity.2.The methylation of OGG1 was regulated by the interaction of H19 and SAHH.3.OGG1 methylation regulated by H19/SAHH plays an important role in the oxidative DNA damage induced by Ba P exposure.