Effect Of Dihydroartemisinin On UHRF1 Gene Silencing Or Overexpression In Prostate Cancer PC-3 Cells And Underlying Regulatory Mechanism

Objective:To investigate the effects and mechanisms of Dihydroartemisinin（DHA）on Ubiquitin-like with PHD and ring finger domains 1（UHRF1）silencing or overexpression in prostate cancer PC-3 cells.Methods:Lentivirus particles containing UHRF1 or sh RNA-UHRF1 were used to infect prostate cancer PC-3 cells respectively,and cell models with UHRF1 overexpression or interference were constructed.Cell models in each group were treated with different concentrations of DHA（0,25,50and 100μmol/L）.CCK-8 assay,scratch assay and FCM assay were used to detect the proliferation,migration,cell cycle and apoptosis of the above cell models and drug-treated groups.The m RNA and protein expressions levels of UHRF1,DNMT1 and p16^INK4Ain each cell models and drug-treated groups were detected by real-time quantitative PCR,western blot and cellular immunofluorescence staining.Results:1.CCK-8 and scratch assay showed that DHA inhibited the proliferation and migration of PC-3 cells（P<0.05）,and the overexpression of UHRF1 resisted the inhibitory effect of DHA to a certain extent,and the silencing of UHRF1 combined with DHA amplified the inhibitory effect（P<0.05）.2.FCM showed that DHA induced PC-3 cell apoptosis and G1/S phase arrest（P<0.05）.Overexpression of UHRF1 increased the proportion of G2/M phase cells and resisted the pro-apoptotic effect of DHA to a certain extent.Silencing UHRF1 combined with DHA increased the percentage of G1/S phase arrest cells and apoptotic cells（P<0.05）.3.qRT-PCR and western blotting showed that DHA inhibited the expression of UHRF1 and DNMT1,and up-regulated the expression of p16^INK4A（P<0.05）.Overexpression of UHRF1 resisted the regulation of p16^INK4Aby DHA to some extent,but enhanced the inhibitory effect of DHA on DNMT1 at protein level.Silencing UHRF1 combined with DHA further increased the expression of p16^INK4A（P<0.05）.4.Cell immunofluorescence test showed that overexpression of UHRF1 significantly reduced the protein expression levels of p16^INK4Ain the nucleus of PC-3 cells.Silencing of UHRF1 promoted the protein expression levels of p16^INK4Ain the nucleus,and further restored the expression of p16^INK4Aafter DHA treated（P<0.05）.Conclusion:UHRF1 may promote the proliferation and metastasis of PC-3 cells by inhibiting the expression of p16^INK4A.DHA can effectively inhibit the proliferation and metastasis of PC-3 cells and promote cell apoptosis,which may be related to the down-regulation of UHRF1 to restore the expression of p16^INK4A,thus exerting the anti-cancer effect.These findings above suggest that the UHRF1/DNMT1 axis may be an important pathway for DHA to regulate PC-3 cells proliferation.