Regulation Of CDC20 Transcription By LEF1 And Its Effects On Proliferation,apoptosis And Cycle Of Liver Cancer Cells

[Objective] To investigate whether lymphoid enhancer-binding factor 1(LEF1)regulats the expression of cell division cycle protein 20(CDC20)in liver cancer cells,and to study its effects on proliferation,apoptosis and cell cycle of hepatoma cells.[Methods](1)In three kinds of cultured human liver cancer cell lines Huh-7,HepG2 and SMMC-7721,real time PCR and Western blot respectively were applied to detect LEF1 and CDC20 expression;(2)Chromatin immunoprecipitation(ChIP)detected transcription factor LEF1 and CDC20 promoter region binding;(3)Dual luciferase reporter gene detection system was conducted to assess the changes of CDC20 promoter transcriptional activity after abnormal expression of LEF1;(4)Using bioinformatics method predicted the sites of transcription factor LEF1 binding to CDC20 promoter region.On the basis of them,we designed and synthetised relative probes,used EMSA to detect and verify the specific binding site;(5)Constructed lentiviral vectors of LEF1 overexpression and interference and the corresponding empty vectors were respectively transfected into Hep G2 cells with relatively low expression of LEF1 and into Huh-7 cells with relatively high expression of LEF1.The transfection efficiency was observed by fluorescence microscopy.Real time PCR and Western blot were used to detect the expression of LEF1 and CDC20 after transfection;(6)Ability of cell growth was examined by clone formation assay after transfection;(7)Cell apoptosis after transfection was detected by the AnnexinV-PE/7AAD method;(8)The changes of cell cycle after transfection were test by DAPI staining.(9)The HepG2 cells with stable overexpression of LEF1 interfered with CDC20 was detected wheather cell proliferation,apoptosis and cell cycle were affected.[Results](1)The relative expression of LEF1 in the three different hepatoma cell lines was the highest in Huh-7,followed by SMMC-7721,while the expression in HepG2 was the lowest.(2)ChIP results showed that the transcription factor LEF1 could bind to the CDC20 DNA promoter in HepG2 and Huh-7 cells.(3)The relative activity of luciferase of CDC20 reporter gene after transfection of pcDNA3.1-LEF1 was significantly increased(p < 0.05).(4)Online PROMO software predicted that there were 5 LEF1 binding sites called Site1-5,in the upstream 1.6kb region of CDC20 transcription initiation that might bind to LEF1.The results showed that Site1-2 interacted LEF1 corresponding to probe1 in Probe1-4.(5)Real time PCR and Western blot confirmed mRNA and the protein expression level of LEF1 and CDC20 in the cells transfected LV-LEF1 cDNA were significantly higher than that in negative control group(P < 0.05),but the expression level of mRNA and protein of LEF1 and CDC20 in LV-LEF1 siRNA cells was significantly lower than that in negative control group(P < 0.01).(6)Compared with the control group,the proliferation of HepG2 cells increased significantly after LEF1 overexpression(P < 0.05),whereas the proliferation of interfered Huh-7 cells decreased significantly(P < 0.05).(7)The apoptosis of HepG2 cells decreased significantly(P < 0.01)after overexpression of LEF1,and the apoptosis of Huh-7 cells after interference increased significantly(P < 0.01).(8)The G2/M phase cells increased significantly in HepG2 after LEF1 overexpression(P < 0.01),while the G2/M phase cells of Huh-7 cells decreased significantly(P < 0.01).(9)The HepG2 cells with stable overexpression of LEF1 interfered with CDC20 was found cell proliferation decreased(P<0.01),apoptosis increased(P<0.01)and cell cycle changed(P<0.01).[Conclusion](1)Transcription factor LEF1 combining CDC20 upregulates the expression of CDC20 in hepatoma cells;(2)The effect of LEF1 on CDC20 can affect the biological behavior of hepatoma cells,which promote cell proliferation and inhibit cell apoptosis and cause the percentage of cells in G1/S phase decreasing and G2/M phase increasing,significantly influence cell cycle.