5-Aza-CdR Inhibits Expression Of Apo(a) Through Downregulating FXR Gene Methylation

Object:To analyze the DNA demethylation of5-Aza-CdR effect on the expressionregulation of apo(a) in HepG2cells, and explore its mechanism.Method:HepG2cell,an high apo(a) expression cell lines,were selected for ourstudy,①HepG2cells were treated with different concentrations(0,10,20,40,80μmol/L)and different time(12,24,48,72,96hr),then detected the relative survival ratio ofHepG2cells by MTT method,Selected the appropriate drug concentration and effecttime.②Detected the expression of apo(a) and FXR in HepG2cells treated with5-Aza-CdR by western blot,and the expression of apo(a)mRNA and FXRmRNA byRT-PCR.③Detected the DNA methylation status of FXR gene promoter in different5-Aza-CdR concentration by BSP（Bisulfite Sequencing PCR）.Result：①MTT results show that：compared with control group，at different timepoint（12hr、24hr、48hr、72hr），the survival ratio of HepG2cells had no significantdifference in group10μmol/L,20μmol/L and40μmol/L (P＞0.05).but in group of20μmol/L96hr,40μmol/L96hr,80μmol/L24hr,80μmol/L48hr,80μmol/L72hr and80μmol/L96hr,the survival ratio of HepG2cells had obviously significantdifference.we chose0μmol/L to40μmol/L as safe concentration range,and selected0hr to72hr as appropriate time.②Western blot results show that:compared withcontrol group,the expression of apo(a) were downregulated by5-Aza-CdR with doseand time dependent,while the expression of FXR were upregulated by5-Aza-CdRwith does and time dependent,and40μmol/L and72hr group were changedobviously(P＜0.05).③RT-PCR results show that:compared with control group,theexpression of apo(a)mRNA were downregulated by5-Aza-CdR with dose and timedependent,while the expression of FXRmRNA were upregulated by5-Aza-CdR withdoes and time dependent,and40μmol/L and72hr group were changed obviously(P＜0.05).④BSP results shows that:with the increase of concentration of5-aza-CdR,themethylation status of FXR gene promoter gradually reduced,and the methylation ratio of control group was58.3%,the40μmol/L group was8.3%.Conclusion:1.the expression of apo(a) in HepG2cells were inhibited by5-Aza-CdRwith dose and time dependent;2.5-Aza-CdR promote the expression of FXR though the demethylationeffect,then downregulate the expression of apo(a).