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慢病毒介导的过表达miRNA-663对膀胱癌细胞功能学影响
The effect of Lentivirus-mediated overexpression miRNA-663 on biological behaviors in human bladder cancer cell lines in vitro
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DOI:
作者:
宋瑞祥,曾蜀雄,赵俊杰
SONG Ruixiang,ZENG Shuxiong,ZHAO Junjie
作者单位:
上海长海医院泌尿外科泌尿外科
Department of urology,Changhai Hospital,Second Military Medical University, Shanghai
关键词:
慢病毒;膀胱癌;EJ和5637;小RNA-663
lentiviral vector; bladder cancer; EJ and 5637 cells; miRNA-663
摘要:
目的 探讨负载miRNA-663(miR-663)的慢病毒(Lentivirus,LV)上调miR-663对膀胱癌细胞系EJ和5637功能学的影响。 方法 将负载miR-663慢病毒转染人膀胱癌细胞系EJ和5637。各细胞系设为干扰组(LV-miR-663组,LV组)、阴性对照组(negative control组,NC组)和空白对照组(blank组,B组);转染72h后于荧光显微镜下观察转染效率;分别采用real-time PCR方法检测miR-663的表达量;采用CCK8法检测细胞增殖生长情况,;采用细胞划痕实验检测miR-663对膀胱癌细胞迁移能力的影响;采用Transwell小室侵袭实验检测miR-663对膀胱癌细胞侵袭能力的影响。 结果 EJ和5637细胞系中,与B组和NC组相比,LV组转染膀胱癌细胞后可显著上调mi-63的表达。CCK8实验显示LV组细胞增值能力较B组和NC组降低,EJ细胞自第4 天期和5637细胞自第3天起细胞生长受抑制(P<0.05)。划痕实验显示,LV组细胞迁移修复能力明显低于B组和NC组(P<0.05)。Transwell实验显示,LV组癌细胞侵袭能力明显减弱(P<0.05)。 结论 miR-663体外可显著抑制膀胱癌EJ和5637细胞增值、迁移及侵袭能力,为miR-663作为膀胱癌的治疗新靶点提供了实验依据。
Objective: To investigate the effect of lentivirus--mediated miRNA-663 on biological behaviors in human bladder cancer cell lines EJ and 5637 in vitro. Methods: The human bladder cancer cell lines(EJ and 5637) were infected with LV-miR-663.The groups were arranged as cells without treatment(Blank group, B),cells transfected with lentivirus empty vector(negative control group,NC ) and cells transfected with lentivirus-mediated-miR-663(lentivirus miR-663 group,LV). After transfecting for 72h, transfection efficiency was determined by fluorescence microscopy. Real time fluorescene quantitation polymerase chain reaction(real-time PCR) was used to determine the expressions of miR-663 in three groups of two cell lines.The influence of miR-663 on bladder cancer cells proliferation were determined by CCK-8, and the migration ability was determined by wound healing test. The invasion was detected by Transwell test. Results: After infected with LV-miR-663, the expression of miR-663 in EJ and 5637 cells was up-regulated significantly compared with B group and NC group. The proliferation of LV groups were also markedly suppressed in CCK-8(P<0.05). The number of invasive cells that migrated through the chamber decreased(P<0.05). The migration abilities of LV groups decreased in wound healing tests than B groups and NC groups both cell lines. Conclusion miR-663 can effectively suppress the proliferation ,migration and invasion of human bladder cancer cell lines EJ and 5637 in vitro. These findings could provide some basis for miR-663 maybe as a therapeutic target for bladder cancer.
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