The Effects Of GANT61 On Notch Signaling Pathway In Multiple Myeloma

Objective:To explore the effects of GANT61 on Notch signaling pathways in vitro on RPMI8226 and U266,and to explore the relationship between Notch pathway and Hedgehog pathway in multiple myeloma cell lines,and to provide experimental data for the clinical treatment of multiple myeloma.Methods:1.Multiple myeloma cell lines RPMI8226 and U266 were cultured in vitro,the experimental group: blank control group,2.5,5,10umol/L GANT61 group.2.CCK-8 method was used to test the proliferation inhibition rate treated with different concentrations of GANT61 on different time in RPMI8226 and U266 cell lines.3.Real-time quantitative PCR(Real-time q PCR,RT-qPCR)was used to detect the mRNA expression of Notch pathway receptor Notch1,ligand Jagged1,Jagged2 and target gene Hes1.4.Western blot was used to detect the protein expression of Notch pathway receptor Notch1,ligand Jagged1,Jagged2 and target gene Hes1.Results:1 CCK8 method to detect the proliferation inhibition rate of GANT61 on MM cell linesThe inhibitory rates of RPMI8226 and U266 cell lines treated with different concentrations of GANT61(2.5,5,10,20,30,40,50umol/L)for 18 h,24h and 36 h were shown in Fig 1,2 and Tab 1 and 2.With the increase of GANT61 concentration and the prolongation of treatment time,the proliferation inhibition rate increased gradually.For example,in RPMI8226 cell line,the inhibitory rates of 2.5,5,10,20,30,40 and 50umol/L GANT61 were(0.13±0.02)%,(0.19±0.04)%,(0.30±0.01)%,(0.45±0.00)%,(0.58±0.02)%,(0.63±0.02)%,(0.70±0.02)%,while in U266,the inhibition rates were(0.16±0.04)%,(0.25±0.02)%,(0.42±0.02)%,(0.53±0.01)%,(0.63±0.01)%,(0.71±0.05)%,(0.73±0.01)%.The comparison of the same concentration at different time was shown in Table 1 and Table 2.And in RPMI8226 cell line treated with 5umol/L GANT61 at 18 h,24h and 36 h,the inhibition rates were(0.15±0.03)%,(0.19±0.04)%,(0.22± 0.04)%,and in U266,were(0.23±0.00)%,(0.25±0.02)%,(0.36±0.04)%.At the same time,the difference between GANT61 and the bank control was statistically significant(P<0.05).The comparison result was shown in Tab1,2;At the same concentration of GANT61,the comparison of different time was shown in Table 1,2.According to the experimental result,the concentration of GANT61 was selected 2.5,5,10umol/L,and the time was selected 18 h,24h and 36 h.2 The influence of GANT61 on the expression of Notch1 in RPMI8226 and U266 cell2.1 GANT61 down-regulated the mRNA expression of Notch1 in RPMI8226 and U266 cell lines.The mRNA expression of Notch1 decreased with the increase of GANT61 concentration,and the difference was statistically significant(P<0.05).The mRNA expression of Notch1 decreased gradually with the prolongation of time,the difference was statistically significant(P<0.05).2.2 GANT61 down-regulated the protein expression of Notch1 in RPMI8226 and U266 cell lines.The protein expression of Notch1 decreased with the increase of GANT61 concentration,and the difference was statistically significant(P<0.05).The protein expression of Notch1 decreased gradually with the prolongation of time,the difference was statistically significant(P<0.05).3 The influence of GANT61 on the expression of Jagged1 and Jagged2 in RPMI8226 and U266 cell3.1 GANT61 down-regulated the mRNA expression of Jagged1 and Jagged2 in RPMI8226 and U266 cell lines.The mRNA expression of Jagged1 and Jagged2 decreased with the increase of GANT61 concentration,and the difference was statistically significant(P<0.05).The mRNA expression of Jagged1 and Jagged2 decreased gradually with the prolongation of time,the difference was statistically significant(P<0.05).3.2 GANT61 down-regulated the protein expression of Jagged1 and Jagged2 in RPMI8226 and U266 cell lines.The protein expression of Jagged1 and Jagged2 decreased with the increase of GANT61 concentration,and the difference was statistically significant(P<0.05).The protein expression of Jagged1 and Jagged2 decreased gradually with the prolongation of time,the difference was statistically significant(P<0.05).4 The influence of GANT61 on the expression of Hes1 in RPMI8226 and U266 cell4.1 GANT61 down-regulated the mRNA expression of Hes1 in RPMI8226 and U266 cell lines.The mRNA expression of Hes1 decreased with the increase of GANT61 concentration,and the difference was statistically significant(P<0.05).The mRNA expression of Hes1 decreased gradually with the prolongation of time,the difference was statistically significant(P <0.05).4.2 GANT61 down-regulated the protein expression of Hes1 in RPMI8226 and U266 cell lines.The protein expression of Hes1 decreased with the increase of GANT61 concentration,and the difference was statistically significant(P<0.05).The protein expression of Hes1 decreased gradually with the prolongation of time,the difference was statistically significant(P<0.05).Conclusion:1 GANT61 inhibited the proliferation of multiple myeloma cell lines RPMI8226 and U266,and the proliferation inhibition rate increased gradually in a time-dose dependent manner.2 GANT61 can inhibit the mRNA and protein expression of Notch1,Jagged1,Jagged2 and target gene Hes1 in Notch signaling pathway of RPMI8226 and U266 cell lines,which is in a time-dose dependent manner.