Study The Effect Of ShRNA NOTCH1on Silencing NOTCH1Gene By Eukaryotic Vector In Multiple Myeloma

Objective: To study the effect of shRNA NOTCH1on silencing NOTCH1geneon mutiple myenoma cell line, RPMI8226cells, we transfect NOTCH1shRNA toRPMI8226cells and investigate cell growth, apoptosis and secretion of IL-6.Methods：⑴Four pairs ofhairpin-like oligonucleotide sequences specific forhuman NOTCH1gene were synthesized, and screened. NOTCH1shRNA wastransfected into RPMI8226cells by LipofectamineTM2000. The optimal sequenceswas screened by RT-PCR.⑵Cell growth affected by NOTCH1shRNA wasdetermined by MTT. Cell apoptosis and cell cycle were measured by Flow cytometry.The expression of notch-1, P21and P27were detected by Western blot.⑶IL-6secretion affected by NOTCH1shRNA was detected by enzyme-linkedimmunosorbent assay (ELISA).Results:⑴The optimal sequenceof NOTCH1shRNA is Sense5’-CACCGGGACATCACGGATCATATGGTTCAAGAGACCATATGATCCGTGATGTCCCTTTTTTG-3’,Anti-sense5’-GATCCAAAAAAGGGACATCACGGATCATATGGTCTCTTGAACCATATGATCCGTGATGTCCC-3’.⑵The cell growthin NOTCH1shRNAwas (49.09±2.31)%, Neg-shRNA was (91.73±2.67)%, blank was (98.10±0.41)%(p<0.05). The cell phase in G0/G1was (66.23±3.71)%,(42.27±3.65)%,(40.70±0.92)%respectively in NOTCH1shRNA, Neg-shRNA and blank (p<0.05). The Sphase was (31.03±1.49)%,(56.53±1.76)%,(51.83±1.45)%in NOTCH1shRNA,Neg-shRNA and blank respectively (p<0.05)．Compared to control, the expression ofnotch-1was decreased, fell by44.54%. The expression of P27and P21wereincreased for1.75folds and1.96folds by transfected NOTCH1shRNA. The cellapoptotic rate was (46.67±1.31)%,(1.37±0.63)%,(0.85±0.43)%in NOTCH1 shRNA, Neg-shRNA and Blank respectively. The cell apoptosis rate in NOTCH1shRNA was significantly higher than those in Neg-shRNA or Blank (p<0.05)．⑶ODvalue of IL-6secretion in NOTCH1shRNA transfection was28.18±3.50,Neg-shRNA was167.08±7.97, and blank was185.86±5.61, p<0.05.Conclusions:⑴NOTCH1shRNA could inhibit NOTCH1gene in RPMI8226cells.⑵NOTCH1shRNA inhibites cell growth by blocked Cell cycle in G0/G1phase.It upregulates P21, P27, which might be one of the mechnisms of inhibition of cellproliferation.⑶NOTCH1shRNA induces cell apoptosis in RPMI8226cells.⑷Itdownregulates IL-6secretion. It might be a novel therapeutic target in MM.