胰岛素样生长因子2基因干扰对人膀胱癌细胞HTB-95637细胞周期和增殖的影响

中国临床药理学与治疗学 ›› 2014, Vol. 19 ›› Issue (11): 1233-1237.

胰岛素样生长因子2基因干扰对人膀胱癌细胞HTB-95637细胞周期和增殖的影响

钱苏波¹, 陈牧², 沈海波¹, 齐隽¹

¹ 上海交通大学医学院附属新华医院泌尿外科,上海 200092;
² 上海交通大学医学院,上海 200025

收稿日期:2014-03-04 修回日期:2014-08-08 出版日期:2014-11-26 发布日期:2014-12-09
通讯作者: 齐隽,男,教授,主任医师,博士生导师,研究方向:泌尿系统肿瘤的临床与基础研究。 Tel: 13901817928 E-mail: jasonqi@sh163.net
作者简介:钱苏波,男,博士研究生,研究方向:泌尿系统肿瘤的临床与基础研究。Tel: 13761020575 E-mail: qiansubo@126.com

Effects of shRNA targeting IGF2 gene on the growth and cell cycle of human bladder cancer cells

QIAN Su-bo¹, CHEN Mu², SHEN Hai-bo¹, QI Jun¹

¹ Department of Urology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092, China;
² Shanghai Jiaotong University School of Medicine, Shanghai 200025, China

Received:2014-03-04 Revised:2014-08-08 Online:2014-11-26 Published:2014-12-09

摘要/Abstract

摘要： 目的探讨胰岛素样生长因子2(insulin-like growth factor 2, IGF2)基因干扰对人膀胱癌细胞株HTB-95637细胞周期和增殖的影响。方法以人膀胱癌细胞株HTB-95637为研究对象,针对其IGF2基因序列设计小干扰RNA(small interfering RNA, siRNA),筛选出能高效沉默目标基因的片段序列后,重组其短发夹状RNA(short hairpin RNA, shRNA)腺病毒表达载体,转染人膀胱癌细胞株HTB-95637,筛选出能稳定表达干扰质粒的细胞克隆。采用逆转录聚合酶链反应(RT-PCR)和免疫印迹法(Western blot)分别检测转染后IGF2的mRNA和蛋白表达水平,用四甲基偶氮唑蓝法(MTT法)检测细胞增殖能力,用PI单染流式细胞术分析细胞周期的改变。结果 RT-PCR和Western blot结果显示,shRNA对IGF2在基因和蛋白表达水平均有显著的下调作用。稳定转染IGF2-shRNA的人膀胱癌细胞株HTB-95637的生长速度明显减缓,处于细胞周期S期的细胞比例由 42.0%±3.8%上升至 70.2%±5.2%,而处于G₂/M期的细胞比例由 9.7%±2.4%下降为 1.5%±1.6%。结论靶向IGF2基因的shRNA可抑制膀胱癌细胞株HTB-95637细胞IGF2基因的表达,调控HTB-95637的细胞周期,抑制HTB-95637的细胞增殖,具有潜在的开发应用前景。

关键词: 膀胱癌, 胰岛素样生长因子2, 小干扰RNA, 细胞增殖, 细胞周期

Abstract: AIM: To investigate the effect of short hairpin RNA (shRNA) targeting insulin-like growth factor 2 (IGF2) gene on the growth and cell cycle of human bladder cancer cells. METHODS: Human bladder cancer cells HTB-95637 were available for this study. Some small interfering RNA (siRNA) fragments targeting IGF2 gene were designed, and those could silence the target gene sequence efficiently were chosen. Then, the shRNA recombiant adenovirus expression vectors were structured. These vectors were transfected to HTB-95637 cells subsequently, and the cell clones could express targeting plasmid stably were screened. Afterwards, reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to detect the expression level of IGF2 mRNA and protein, MTT method was used to detect the proliferation of the HTB-95637 cells, and PI single staining flow cytometry was used to analysis the change of cell cycle. RESULTS: The results of RT-PCR and Western blot indicated that the designed shRNA could down-regulate the mRNA and protein expression of IGF2 gene. The growth rate of the HTB-95637 cells transfected with IGF2-shRNA was slower than that in other groups. Compared with other groups, the percentage of cells in S phase increased from 42.0%±3.8% to 70.2%±5.2% and those in G₂/M phase decreased from 9.7%±2.4% to 1.5%±1.6% when transfected with IGF2-shRNA.CONCLUSION: The shRNA targeting IGF2 gene could inhibit the gene expression, regulated the cell cycle and restrained the cell proliferation of human bladder cancer cells.

Key words: bladder cancer, insulin-like growth factor 2 (IGF2), siRNA, cell proliferation, cell cycle

中图分类号:

R965.2

钱苏波, 陈牧, 沈海波, 齐隽. 胰岛素样生长因子2基因干扰对人膀胱癌细胞HTB-95637细胞周期和增殖的影响[J]. 中国临床药理学与治疗学, 2014, 19(11): 1233-1237.

QIAN Su-bo, CHEN Mu, SHEN Hai-bo, QI Jun. Effects of shRNA targeting IGF2 gene on the growth and cell cycle of human bladder cancer cells[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2014, 19(11): 1233-1237.

参考文献 19

[1]	Kaneda A, Wang CJ, Cheong R, et al.Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk[J]. Proc Natl Acad Sci U S A, 2007, 104(52):20926-20931.
[2]	Oda H, Kume H, Shimizu Y, et al.Loss of imprinting of igf2 in renal-cell carcinomas[J]. Int J Cancer, 1998, 75(3):343-346.
[3]	Sakatani T, Kaneda A, Iacobuzio-Donahue CA, et al.Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice[J]. Science, 2005, 307(5717):1976-1978.
[4]	Cui H, Cruz-Correa M, Giardiello FM, et al.Loss of IGF2 imprinting: a potential marker of colorectal cancer risk[J]. Science, 2003, 299(5613):1753-175.
[5]	Seo JH, Park BC.Expression of insulin-like growth factor II in chronic hepatitis B, liver cirrhosis, and hepatocellular carcinoma[J]. Gan To Kagaku Ryoho, 1995, 22(Suppl 3):292-307.
[6]	Wu MS, Wang HP, Lin CC, et al.Loss of imprinting and overexpression of IGF2 gene in gastric adenocarcinoma[J]. Cancer Lett, 1997, 120(1):9-14.
[7]	Nakagawa H, Chadwick RB, Peltomaki P, et al.Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer[J].Proc Natl Acad Sci U S A, 2001, 98(2):591-596.
[8]	甘德芳, 高天翼, 王书奎, 等. 结直肠癌患者中IGF2 LOI 以及表达的研究[J]. 现代生物医学进展, 2013,13(11): 2090-2092.
[9]	Siegel R, Ma J, Zou Z, et al.Cancer statistics, 2014[J]. CA Cancer J Clin, 2014, 64(1): 9-29.
[10]	Byun HM, Wong HL, Birnstein EA, et al.Examination of IGF2 and H19 loss of imprinting in bladder cancer[J]. Cancer Res, 2007, 67(22):10753-10758.
[11]	Elkin M, Shevelev A, Schulze E, et al.The expression of the imprinted H19 and IGF-2 genes in human bladder carcinoma[J]. FEBS Lett, 1995, 374(1):57-61.
[12]	Pollak M.Insulin and insulin-like growth factor signalling in neoplasia[J]. Nat Rev Cancer, 2008, 8(12):915-928.
[13]	高军, 苏晖, 黄伟, 等. 膀胱癌组织中IGF-2和H19的基因印迹状态及其临床意义[J]. 中国医药导刊, 2010 ,12(4): 585-587.
[14]	高军, 苏晖, 黄伟, 等. 膀胱癌中IGF-2和H19基因特异表达模式及印记缺失分析[J]. 牡丹江医学院学报, 2010, 31(5): 13-16.
[15]	Ariel I, de Groot N, Hochberg A. Imprinted H19 gene expression in embryogenesis and human cancer: the oncofetal connection[J]. Am J Med Genet, 2000, 91(1):46-50.
[16]	Banet G, Bibi O, Matouk I, et al.Characterization of human and mouse H19 regulatory sequences[J]. Mol Biol Rep, 2000, 27(3):157-165.
[17]	Byun HM, Wong HL, Birnstein EA, et al.Examination of IGF2 and H19 loss of imprinting in bladder cancer[J]. Cancer Res, 2007, 67(22):10753-10758.
[18]	安立峰, 董震. RNA干扰--肿瘤研究的新工具[J]. 中华肿瘤杂志, 2005, 27(7): 385-388.
[19]	赵晓艳, 孙立新. RNA干扰技术在肿瘤治疗中的研究进展[J]. 国际肿瘤学杂志, 2014, 41(3): 177-179.