Quercetin Demethylation And Its Interaction With Benzopyrene (BaP) On Cell Cycle And Apoptosis

Objective: To detect the methylation of FHIT and CDH13 genes in Mongolian and Han liver cancer patients in Inner Mongolia,analyze the relationship between methylation of FHIT ?CDH13 genes and liver cancer,and determine whether there are national differences in methylation of FHIT and CDH13 genes.Demethylation and Interaction with Benzopyrene(Ba P)on Cell Cycle and Apoptosis.Methods: Methylation specific polymer chain reaction(MSP)was used to detect the methylation of FHIT and CDH13 gene promoter regions in Mongolian and Han healthy population and liver cancer patients with liver cancer in Inner Mongolia;MSP method was used to detect the methylation of FHIT and CDH13 genes in Hep G2 cells at different concentrations of quercetin;Real-time fluorescence quantitative polymerase chain reaction(q PCR)and Western blot were used to detect FHIT,CDH13 gene,DNA methylation transferase 1(DNMT1),and transcription factor Sp1 after quercetin at different concentrations on Hep G2 cells expression of m RNA and protein;Flow cytometry and Hochest33342 were used to detect the interaction of quercetin and benzopyrene(Ba P)on cell cycle and apoptosis.Results:(1)The methylation rates of FHIT and CDH13 genes in Mongolian liver cancer patients were 72.00%(36/50)and 64.00%(32/50),while the methylation rates of FHIT and CDH13 genes in Mongolian healthy people were 34.00%(17/50)and 32.00%(16/50),the difference in methylation rates of FHIT and CDH13 genes between Mongolian liver cancer patients and healthy people was statistically significant(P<0.05);(2)The methylation rates of FHIT and CDH13 genes in Han liver cancer patients were 68.00%(34/50)and 72.00%(36/50),and the methylation rates of FHIT and CDH13 genes in Han healthy population were 30.00%(15/50)and 22.00%(11/50).The difference was statistically significant(P<0.05);(3)Comparing the methylation rates of FHIT and CDH13 genes between Mongolian and Han liver cancer patients and healthy people,there was no national difference and no statistical significance(P>0.05);(4)Comparing the methylation rates of FHIT and CDH13 genes in healthy people of Mongolian and Han ethnic groups,it was found that there were no ethnic differences and no statistical significance(P>0.05);(5)MSP test results showed that compared with the control group,as the concentration of quercetin increased,the brightness of the methylated bands(M bands)of FHIT and CDH13 genes gradually decreased,and unmethylated bands(U bands)appeared.Specific amplification and brightness gradually increase,quercetin has a certain degree of reversal effect on FHIT,CDH13 gene methylation;(6)The results of q PCR and Western blot showed that compared with the control group,the m RNA and protein expressions of FHIT and CDH13 genes increased gradually with the increase of quercetin concentration,and the expression results of FHIT and CDH13 in the quercetin medium and high dose groups Compared with the control group,the differences were statistically significant(P<0.05,P<0.01).With the increase of quercetin concentration,the m RNA and protein expression of DNMT1 and Sp1 gradually decreased,and the differences were statistically significant compared with the control group.(P<0.05,P<0.01);(7)Flow cytometry results showed that quercetin low dose + Ba P group,quercetin medium dose + Ba P group,quercetin high dose + Ba P group compared with quercetin low,medium and high dose groups.The percentages of both phase S and G2/M phase increased,which enhanced the blocking effect on phase S and G2/M phase,and the high-dose group was statistically significant(P<0.05);Hochest33342 staining and flow cytometry showed that as the concentration of quercetin increases,its apoptosis-inducing effect gradually increases.The apoptotic rate is: low-dose group(38.43�0.77%),medium-dose group(42.20�0.57%),and high-dose group(59.76�0.58%)with apoptosis of quercetin low dose + Ba P group(36.83�0.47%),quercetin medium dose + Ba P group(38.18�0.56%),quercetin high dose + Ba P group(43.43�0.46%)Compared with quercetin alone,the apoptosis rate of Hep G2 cells was higher than that of Hep G2 cells at the same time,and there were statistical differences in low,medium and high dose groups(P<0.05).Conclusion: FHIT and CDH13 genes are hypermethylated in liver cancer patients,and there is no ethnic difference between Mongolian and Han nationalities in Inner Mongolia;Quercetin has a demethylation effect,and its possible mechanism is to inhibit Sp1,thereby inhibiting the expression of DNMT1,when the activity of DNMTs is inhibited or the concentration is too low,the original methylation state cannot be maintained,resulting in FHIT,CDH13 gene demethylation;Both quercetin and Ba P can block the Hep G2 cell cycle,and they have a synergistic effect;For apoptosis,both Ba P and quercetin can induce apoptosis in Hep G2 cells,but when both act on Hep G2 cells at the same time,the percentage of apoptosis decreases and both have an antagonistic effect.