中国癌症杂志 ›› 2019, Vol. 29 ›› Issue (3): 193-200.doi: 10.19401/j.cnki.1007-3639.2019.03.006

• 论著 • 上一篇    下一篇

RNAi沉默rictor基因表达抑制人膀胱癌T24细胞体内外生长

何春锋1,张青川1,刘 永2   

  1. 1. 上海中医药大学附属普陀医院泌尿外科,上海 200062 ;
    2. 上海交通大学附属第一人民医院泌尿外科,上海 200080
  • 出版日期:2019-03-30 发布日期:2019-04-26
  • 通信作者: 刘 永 E-mail: koucai@medmail.com.cn

Silencing rictor gene by RNA interference inhibits cell growth of human bladder cancer T24 in vitro and in vivo

HE Chunfeng1, ZHANG Qingchuan1, LIU Yong2   

  1. 1. Department of Urology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China; 2. Department of Urology, Shanghai General Hospital, Shanghai 200080, China
  • Published:2019-03-30 Online:2019-04-26
  • Contact: LIU Yong E-mail: koucai@medmail.com.cn

摘要: 背景与目的:膀胱癌是泌尿系统高发肿瘤,浅表性膀胱癌发展成为浸润性膀胱癌后,预后较差。该研究利用RNA干扰(RNA interference,RNAi)技术沉默rictor基因后,观察其对人膀胱癌T24细胞在体内外生长的影响。方法:构建3个rictor-RNAi表达载体(1#、2#和3#)和1个阴性对照载体。采用瞬时转染和稳定转染技术筛选出干扰效果明显的单克隆稳定细胞株。应用细胞计数试剂盒(cell counting kit-8,CCK-8)实验、细胞划痕试验、流式细胞术(flow cytometry,FCM)、裸鼠皮下移植瘤模型研究抑制rictor基因后对T24细胞生长的影响。结果:成功构建rictor-RNAi表达载体和阴性对照载体,瞬时转染T24细胞后,2#蛋白表达量下降最明显。利用2#载体稳定转染T24细胞,筛选2#-1、2#-2两株单克隆细胞。2#-1组和2#-2组细胞较空白组细胞生长减慢(P<0.05)。细胞划痕实验表明,2#-1组和2#-2组细胞的迁移速度也较空白组明显降低(P<0.05)。与空白组和阴性对照组相比,2#-1组和2#-2组G1期细胞比例增加(P<0.05),细胞死亡率增加(P<0.05)。裸鼠皮下移植瘤模型显示,移植瘤的平均体积,2#-1组和2#-2组明显低于空白组和阴性对照组(P<0.001)。结论:成功构建rictor-RNAi稳定表达载体,可有效抑制rictor基因表达。抑制rictor基因表达能显著抑制T24细胞增殖,促进肿瘤细胞死亡,诱使细胞停滞在G1期,并能有效抑制裸鼠移植瘤的生长。RNAi抑制rictor基因的治疗策略可能为膀胱癌的治疗提供新的思路。

关键词: RNA干扰, Rictor基因, 膀胱癌, 抑制

Abstract: Background and purpose: Bladder cancer is a common tumor of urinary system, and the prognosis is poor when superficial bladder cancer develops into invasive bladder cancer. The study aimed to observe the effects of silencing rictor gene using RNA interference (RNAi) on the growth of human bladder cancer T24 cells in vitro and in vivo. Methods: Three rictor-RNAi expression vectors (1#, 2# and 3#) and one negative control vector were constructed. Monoclonal stable cells with significant interference effects were screened by transient transfection and stable transfection techniques. The effects of inhibition of rictor gene expression on the growth of T24 cells were studied using cell counting kit-8 (CCK-8) assay, cell scratch assay, flow cytometry (FCM) and subcutaneous tumor formation in nude mice. Results: We successfully constructed three lines of rictor-RNAi plasmid vectors and one line of negative control vector. 2# vector had the most effective RNAi. 2#-1 and 2#-2 single cell clones were selected after stable transfection with 2# vector. The cell proliferation inhibition rates of 2#-1 group and 2#-2 group were much higher compared with blank control group (P<0.05). The migration speed of 2#-1 group and 2#-2 group also became slower obviously (P<0.05). Compared with blank control group, cell cycle analysis showed that 2#-1 group and 2#-2 group induced accumulation of cells in G1 phase (P<0.05), and cell death analysis showed that 2#-1 group and 2#-2 group induced cell death (P<0.05). The xenograft tumor formation in nude mice showed that the average volumes of cell implantation tumor in 2#-1 and 2#-2 groups were significantly lower compared with the blank group and the negative control group (P<0.001). Conclusion: The rictor-RNAi expression vectors were constructed successfully, and they can reduce rictor gene expression effectively. Inhibition of rictor gene expression can significantly suppress the proliferation of T24 cells, promote tumor cell death, induce G1 phase cell cycle arrest, and effectively inhibit the growth of xenograft tumor in nude mice. The therapeutic strategy of RNAi silencing rictor gene may provide new ideas for the treatment of bladder cancer.

Key words: RNA interference, Rictor gene, Bladder cancer, Inhibit