Inhibition Of UHRF1 Gene Silencing On Invasion And Migration Of Human Lung Adenocarcinoma A549 Cells And Its Effect On Cell Invasion And Migration

objective:UHRF1?Ubiquitin-like with PHD and RING Fing er domains 1?is one of the main members of ubiquitin-like protein famil y containing PHD and ring finger region?UHRF?,and is an important e pigenetic regulator regulating methylation of promoter region of tumor su ppressor gene.By maintaining the methylation of the promoter region of t umor suppressor gene?TSG?,UHRF1 is closely related to the occurrence,development,treatment and prognosis of tumor.At present,there is no re port on the effect and mechanism of UHRF1 gene silencing on invasion and migration of human lung adenocarcinoma A549 cells.The purpose o f this study was to observe the effect of lentivirus-mediated UHRF1 gene silencing on the invasion and migration of human lung adenocarcinoma A549 cells,and to explore its possible regulatory mechanism and provide a new theoretical basis and method for the treatment of non-small cell lu ng cancer.Methods:The A549 cells were seeded in a culture flask cont aining an appropriate amount of RPMI 1640 medium containing 10%fet al calf serum,and cultured in an incubator at 37?in a 5%CO ₂ atmos phere.Cells in the logarithmic growth phase with good growth were used for subsequent experiments.The cell density was adjusted to 5×10⁴/mL one day before transfection and inoculated into 96-well plates at 100?L/well.The experiment was divided into three groups[negative control grou p?NC group?without transfection,negative control group?empty group?transfected with empty vector LV-NC-GFP,and transfected LV-UHRF1-sh RNA-GFP#?39-1?UHRF1 interference group?silent group?].Transwell tes t was used to count the number of cells migrating to the submicroporous layer under inverted microscope,and the number of cells?number/HP?was recorded.Scratch width was observed at 0 h and 24 h by cell scrat ch test,and the relative width of 24 h scratch was recorded.The mRNA and protein expression levels of UHRF1,EMT-related molecules?E-Cad herin,N-Cadherin,Vimentin?in each group were detected by RT-PCR and Western blotting,respectively.Methylation-specific PCR was used to detect the methylation degree of the promoter regions of KISS1 and PAX5 ge nes in cells.All the above experiments were repeated three times and av eraged.Results:1.Lentiviral LV-UHRF1–shRNA-GFP#?39-1?signifi cantly silenced the expression of UHRF1 mRNA and protein after transfe ction into A549 cells,and the difference was statistically significant?P<0.01?.2.Cell invasion and migration ability:After UHRF1 gene silencing,th e number of transmembrane cells in the silencing group was significantly lower than that in the control group?P<0.01?;after UHRF1 gene silencin g,the relative width of scratches at 24 h was significantly larger than th at in the control group,and the difference was statistically significant?P<0.01?.3.Degree of methylation of KISS1 and PAX5:After the silencing of UHRF1 gene,the methylation degree of KISS1 and PAX5 promoter region in silenced group was significantly lower than that in control grou p?P<0.01?.4.Expression of EMT-related molecules:After silencing U HRF1 gene,the expression of N-Cadherin and Vimentin mRNA and prot ein was decreased,and the expression of E-Cadherin mRNA and protein was increased.The difference was statistically significant compared with t he control group?P<0.01?.Conclusion:1.Lentivirus-mediated shRNA sil encing UHRF1 gene expression can inhibit the invasion and migration of A549 cells.2.Silencing UHRF1 gene can reduce the methylation degre e of KISS1 and PAX5 gene promoter regions in A549 cells and inhibit t he process of epithelial-mesenchymal transition?EMT?.3.Silencing the exp ression of UHRF1 gene can inhibit the invasion and migration of A549cells.The mechanism may be related to the reduction of methylation deg ree in the promoter regions of KISS1 and PAX5 genes,thus inhibiting th e process of epithelial-mesenchymal transition.