Effect of Notch-1 Gene Silencing on Biological Behavior of Human Bladder Cancer RT4 Cells in vitro
ZHENG Ke-wen,PAN Yue,HUANG Hang
Department of Urology, the First Af?liated Hospital of Wenzhou Medical University, the First Clinical College of Wenzhou Medical University, Wenzhou (325000), China
Abstract:
Objective To study the effect of Notch- 1 gene silencing on biological behavior such as proliferation, apoptosis, invasion and epithelial-mesenchymal transformation of human bladder cancer RT 4 cells. Methods Notch- 1 shRNA was designed and constructed. RT 4 cells were selected for transfection. The cell experiments were divided into 2 groups: shRNA negative control (shRNA NC) and Notch- 1 shRNA group. After 48 h of cell culture, MTT was used to detect the changes of cell proliferation, Transwell was used to detect the changes of cell invasiveness, ?ow cytometry was used to detect the changes in cell apoptosis. RT- PCR and Western blot were used to detect the changes of apoptosis-related proteins (Bax, Bid and Bcl2) and epithelial mesenchymal transition (EMT) factors (E-cadherin, N-cadherin and Vimentin) at mRNA and protein levels. Results Compared with shRNA NC group, Notch-1 shRNA group showed a significant decrease in cell proliferation rate (t =4.629，P = 0. 010) and invasiveness (t =8.193，P = 0. 001), increased apoptotic rate(t =– 18 . 441 , P < 0 . 001 ), increased mRNA and proteins expression of Bad (t = – 12 . 521 , P < 0.001; t = –6. 983 ,P=0.002), Bid (t= –9.231，P<0.001 ；t=–8.871，P=0.001) and E-cadherin (t= –13.457, P<0.001; t= –5.610,P = 0. 005), and signi?cantly decreased mRNA and proteins expression of Bcl2 (t = 10. 651, P <0.001; t =6.973,P=0.002), N-cadherin (t=6.505, P<0.001; t=10.132,P=0.001) and Vimentin (t=9.135, P<0.001; t=5.630,P = 0 . 005 ) . Conclusion Silencing Notch- 1 expression can inhibit the invasion and migration of human bladder cancer RT 4 cells and promote theirapoptosis. The mechanism may be related to inducing Bad, Bid and E-cadherin and inhibiting the expression of Bcl2, N-cadherin and Vimentin.
