Influence of knocking down BubR1 expression on biochemical behaviors of UMUC3 bladder cancer cells
Objective To investigate the influence of knocking down the expression of BubR1 on the proliferation,migration and invasion of UMUC3 bladder cancer cells.Methods The data of BubR1 mRNA expression in bladder cancer tissue samples and paracancerous tissue samples of 19 patients from TCGA database were collected for comparison.The bladder cancer related gene GSE13507 chip data and clinical data of 165 bladder cancer patients from GEO database were collected.The relative expressions of BubR1 mRNA were compared between bladder cancer tissues and paracancerous tissues,between superficial bladder cancer and invasive bladder cancer,between low-grade bladder cancer and high-grade bladder cancer,and between early bladder cancer and advanced bladder cancer.Taking the relative expression of BubR1mRNA of 165 bladder cancer patients as the boundary value,the overall survival rate and bladder cancer specific survival rate were compared between 78 patients with highly expressed BubR1 mRNA(≥8.68)and 87 patients with lowly expressed BubR1 mRNA(＜8.68).The stable transfection cell line UMUC3 constructed by lentivirus infection method were divided into blank group(transfected with shRNA-NC),transfection group 1(transfected with shRNA BubR1-1),and transfection group 2(transfected with shRNA BubR1-2).After 48-h transfection,the relative expression of BubR1 mRNA was detected by real-time fluorescence quantitative PCR,and the relative expression of BubRl protein was detected by Western blot.The cells in transfection group 2 with the highest knocking down efficiency were used for subsequent experiments.The number of proliferated cells in blank group and transfection group 2 were detected with cytometry by day 1,2,3,4 and 5 after transfection,the scratch width after 12 and 24 h culture was detected by scratch assay,and the cell invasion ability was detected by Transwell chamber assay.Results(1)In 19 patients with bladder cancer,the relative expression of BubR1 mRNA was higher in the bladder cancer tissues(7.00±0.76)than that in the paracancerous tissues(1.22±0.38)(t=6.803,P＜0.001).(2)In 165 patients with bladder cancer,the relative expression of BubR1 mRNA was higher in the bladder cancer tissues(8.68±0.08)than that in the paracancerous tissues(7.83±0.09)(t=5.862,P＜0.001),in the invasive bladder cancer(9.60±0.14)than that in the superficial bladder cancer(8.43±0.09)(t=4.207,P＜0.001),in the high-grade bladder cancer(9.27±0.12)than that in low-grade bladder cancer(8.33±0.09)(t=6.472,P＜0.001),and in the advanced bladder cancer(9.07±0.14)than that in the early bladder cancer(8.45±0.09)(t=3.962,P＜0.001).The overall survival rate and specific survival rate were lower in highly expressed BubR1 group(42.21％,70.50％)than those in lowly expressed BubR1 group(56.78％,83.26％)(P＜0.001).(3)The relative expressions of BubR1 mRNA and protein were lower in transfection group 1(0.47±0.02,0.42±0.04)and transfection group 2(0.19±0.01,0.13±0.02)than those in blank group(1.00±0.08,1.00±0.07)(P＜0.05),and lower in transfection group 2 than those in transfection group 1(P＜0.05).(4)The numbers of cell proliferation by day 4 and 5 after transfection were less in transfection group 2[(16.24±0.57)X 104,(27.74±0.84)X 104]than those in blank group[(18.77±0.41)X104,(33.96±1.66)X 104](P＜0.05).The scratch widths after 12 and 24 h culture were wider in transfection group 2[(1.43±0.02),(0.99±0.03)cm]than those in blank group[(1.29±0.01),(0.42±0.01)cm](P＜0.05).The number of invasive cells was less in transfection group 2(66.67±7.53)than that in blank group(122.30±7.84)(P＜0.05).Conclusions BubR1 is highly expressed in bladder cancer.To knockdown BubR1 can inhibit the proliferation,migration and invasion of UMUC3 bladder cancer cells.BubR1 may be a potential therapeutic target for bladder cancer.
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