天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (12): 1368-1372.doi: 10.11958/j.issn.0253-9896.2015.12.007

• 细胞与分子生物学 • 上一篇    下一篇

TNF-α在结肠癌细胞侵袭和迁移中的作用探讨

赵鹏，陈俊卯，曹文斌，杨光华，虞向阳，刘春辉，郑阳   

  1. 河北唐山，华北理工大学附属医院普外科（邮编063000）
  • 收稿日期:2015-05-13 修回日期:2015-09-15 出版日期:2015-12-15 发布日期:2015-12-11
  • 作者简介:赵鹏（1977），男，副主任医师，博士，主要从事消化道肿瘤的基础与临床研究

Role of TNF-α in promoting migration and invasion of colon cancer cells

ZHAO Peng, CHEN Junmao, CAO Wenbin, YANG Guanghua, YU Xiangyang, LIU Chunhui, ZHENG Yang   

  1. Department of General Surgery, North China University of Science and Technology Affiliated Hospital, Tangshan 063000, China
  • Received:2015-05-13 Revised:2015-09-15 Published:2015-12-15 Online:2015-12-11

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摘要： 目的探讨肿瘤坏死因子（TNF）-α对肿瘤相关钙信号转导蛋白（TROP）-2 表达的影响及其对细胞侵袭和迁移能力的影响。方法以0、10、20、30、50、100、200 μg/L 的TNF-α处理人结肠癌HCT-116 细胞，采用噻唑蓝（MTT）实验检测细胞存活率。采用Western blot 检测TROP-2 的表达量。采用划痕实验和Transwell 实验检测20 μg/L TNF-α处理前后细胞侵袭和迁移能力。借助脂质体Lipofectamine™ 2000 转染TROP-2 siRNA，实时荧光定量 PCR 和Western blot 验证转染后TROP-2 的mRNA 和蛋白水平。划痕实验和Transwell 实验再次检测TNF-α对转染 TROP-2 siRNA 细胞的侵袭和迁移能力的影响。结果0、10、20、30、50 μg/L 的TNF-α处理24 h 和48 h 时细胞存活率差异无统计学意义。100、200 μg/L 与0 μg/L 比较，细胞存活率下降（P＜0.05）。与TNF-α浓度为0 μg/L 比较，10、 20、30、50 μg/L 时TROP-2 的表达量均升高，20 μg/L 时TROP-2 的表达量最高（P＜0.05）;而TNF-α浓度为100、200 μg/L 时TROP-2 的表达量下降（P＜0.05）。划痕实验和Transwell 实验中,TNF-α组24 h 时的细胞划痕愈合率和穿膜细胞数均较对照组增加（P＜0.05）。转染TROP-2 siRNA 后siRNA 组中TROP-2 的mRNA 和蛋白水平均低于正常对照组和阴性对照组（P＜0.05）。此外，siRNA 组24 h 时的细胞划痕愈合率和穿膜细胞数均较正常对照组下降（P＜ 0.05）。结论低浓度炎症因子TNF-α可能通过上调TROP-2 的表达来促进结肠癌HCT-116 细胞的侵袭和迁移。

关键词: 结肠肿瘤, 肿瘤坏死因子α, 肿瘤侵润, 细胞运动, 肿瘤相关钙信号转导蛋白-2

Abstract: Objective To explore the effect of TNF-α on expression of TROP-2 and to explore the role of TROP-2 in the metastasis and invasion of colon cancer HCT-116 cells. Methods HCT-116 cells were cultured and treated with 0, 10, 20, 30, 50, 100 and 200 μg/L TNF-α. Cell viability was assessed by MTT. The expression of TROP-2 was determined by western blot. The effects of 20 μg/L TNF-α on cell migration and invasion were investigated by wound healing assay and Transwell method. Small interfering RNA (siRNA) was used to knock down endogenous TROP-2 expression. The transcrip⁃ tion and translation levels of TROP-2 were detected by qPCR and Western blot respectively. The migratory and invasive ca⁃ pability of HCT-116 cells transfected with TROP-2 siRNA were checked by wound healing assay and Transwell method re⁃ spectively. Results There is no significant change of cell viability between HCT-116 cells treated with 0,10, 20, 30 and 50 μg/L TNF-α, but cell viability of HCT-116 decreased significantly with treatment of 100 μg/L and 200 μg/L TNF-α. Low concentration of TNF-α (≤50 μg/L) led to increase of TROP-2 protein expression that peaks when 20 μg/L TNF-α was add⁃ ed. High concentration of TNF-α (100, 200 μg/L) result in decrease of TROP-2 protein. TROP-2 siRNA significantly downregulated the expression of TROP-2 at both mRNA and protein levels in colon cancer HCT-116 cells. Compared with con⁃ trol group, silencing TROP-2 by TROP-2 siRNA inhibited the migratory and invasive capability of HCT-116 cells. Wound healing rate and the number of transwell cell both decreased in siRNA group compared with those of control group (P < 0.05). Conclusion The mechanism that low concentration of TNF-α promoted HCT-116 cells migration and invasion might be through up-regulating the expression of TROP-2.

Key words: colonic neoplasms, tumor necrosis factor-alpha, neoplasm invasiveness, cell movement, TROP-2