Effect Of MLN4924 On The Invasion And Migration Of HepG2 Cells And The Related Mechanisms

Objective1.Investigate the effect of MLN4924 which is a selective inhibitor of NEDD8-activating enzyme(NAE)on the invasion and migration of HepG2 cells.2.Investigate the molecular mechanisms by which MLN4924 regulates the invasion and migration of HepG2 cells.Methods1.HepG2,Hep3 B and SMMC-7721 cells were intervened with different concentrations(0,1,2,4 and 8 ?mol/L respectively)of MLN4924,then cell counting kit-8(CCK-8)assay was used to detect the proliferation.Apoptosis of HepG2 cells was assessed by apoptosis kits [Annexin V/Propidium Iodide(PI)] and flow cytometry.2.Cell Scratch and Transwell test were used to detect the effect of MLN4924 on invasion and migration.Western Blot method was used to detect the changes of E-cadherin and Vimentin during epithelial-mesenchymal transition(EMT).Results1.By using CCK-8 method,all three hepatoma cell lines were inhabited by MLN4924 and dose-dependent,HepG2 control group showed a sensitivity level towards MLN4924 with IC50 of 3.34?mol/L.Among them,HepG2 was sensitive to MLN4924,the survival rates were 67.09%,63.54%,43.17% and 20.31% at the concentrations of 1,2,4 and 8 ?mol/L,respectively,and the half maximal inhibitory concentration(IC50)values value was 3.34 ?mol/L.After 24 h treatment of HepG2 cells with MLN4924 at a concentration of 1,2,4 and 8 ?mol/L,apoptosis kits [Annexin V/Propidium Iodide(PI)] and flow cytometry detect the rate of apoptosis in each phase,and the total apoptosis rate(%)were(19.36 ± 4.13),(36.50 ± 1.04),(40.03 ± 0.85),(66.53 ± 1.45),which was significantly higher than that of the blank control group(3.50 ± 1.25,P <0.05).2.HepG2 cells treated with MLN4924 at concentrations of 0.5,1,2,and 4 ?mol/L for 12 h and 24 h.Cell scratch test results showed that MLN4924 could significantly inhibit the movement of HepG2 cells,resulting in a decrease in gap closure and with dose and time-dependent.The relative healing rates(%)of wounds at 12 h were(40.00 ± 1.14),(32.54 ± 1.26),(26.05 ± 0.46)and(13.02 ± 1.74),respectively,which were significantly lower than those in the blank control group(53.96 ± 2.37,P <0.05).The relative healing rates(%)of wounds at 24 h were(59.53 ± 1.59),(45.58 ± 2.03),(34.42 ± 2.13)and(20.00 ± 1.59),respectively,which were significantly lower than those in the blank control group(71.16 ± 0.59,P <0.05).The Transwell chambers without Matrigel glue in Transwell experiments demonstrated cellular migration and the Transwell chambers with Matrigel glue demonstrated cellular invasion.The experimental results show that under the influence of different concentrations of MLN4924.The relative migration rates(%)were(28.47 ± 1.13),(10.61 ± 0.66),(4.12 ± 0.14)and(1.21 ± 0.14)respectively,which was significantly lower than that of the blank control group(100.00 ± 5.85,P <0.05).The relative invasion rates(%)were(61.75 ± 10.36),(45.90 ± 9.14),(20.90 ± 4.31)and(12.50 ± 2.20)respectively,which were significantly lower than those in the blank control group(103.60 ± 5.94,P <0.05).Western blot showed that the expression of epithelial marker E-cadherin protein was increased,the relative protein expression was 0.313 ± 0.005,0.594 ± 0.016,0.431 ± 0.002,0.780 ± 0.019,1.000 ± 0.015,and the expression of Vimentin protein was inhibited,Relative protein expression levels were 1.000 ± 0.012,1.158 ± 0.035,1.087 ± 0.076,0.635 ± 0.028,0.351 ± 0.010.It is suggested that MLN4924 may affect the invasion and migration of hepatoma cells through EMT process.Conclusions1.MLN4924 can inhibit HepG2 cells proliferation and induce its apoptosis.2.MLN4924 may inhibit the invasion and migration of HepG2 cells by inhibiting the EMT pathway.