Zhao Peng, Zhang Zhongtao
Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, P.R. China.
Oncol Lett. 2018 Mar;15(3):3820-3827. doi: 10.3892/ol.2018.7735. Epub 2018 Jan 5.
High levels of tumor-associated calcium signal transduction protein (TROP)-2 have been demonstrated to be strongly associated with tumor necrosis factor (TNF)-α levels in colon cancer. In the present study, the effect of TNF-α on the regulation of TROP-2 expression and its effect in colon cancer cell migration and invasion were investigated . TROP-2 protein levels were evaluated in HCT-116 human colon cancer cells cultured with various concentrations of TNF-α using western blot analysis. Changes in the migratory and invasive potential of the cells were evaluated using a wound healing and transwell assay, respectively. Then, TROP-2 expression was downregulated in cells by use of siRNA, and TROP-2 knockdown was confirmed at the mRNA and protein level by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The migration and invasion potential of cells transfected with TROP-2 siRNA was also evaluated. Levels of several mitogen-activated protein kinase proteins, namely p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), were detected in cells treated with TNF-α using western blot analysis. The results demonstrated that TROP-2 protein levels increased in cells treated with lower concentrations of TNF-α, but decreased in cells treated with higher concentrations of TNF-α, compared with untreated control. Maximum TROP-2 levels were observed in cells treated with 20 µg/l TNF-α. Migration and invasion were enhanced in cells treated with 20 µg/l TNF-α. When TROP-2 was silenced in colon cancer cells by siRNA, migration and invasion were significantly decreased compared with control cells. TNF-α stimulation activated the ERK1/2 pathway, but did not significantly affect p38 and JNK phosphorylation levels. Treatment with a specific ERK1/2 inhibitor suppressed the TNF-α-induced upregulation of TROP-2 and the TNF-α-induced colon cancer cell migration and invasion. In conclusion, the present results demonstrated that low concentrations of TNF-α significantly enhanced colon cancer cell migration and invasion by upregulating TROP-2 via the ERK1/2 signaling pathway.
高水平的肿瘤相关钙信号转导蛋白（TROP）-2已被证明与结肠癌中的肿瘤坏死因子（TNF）-α水平密切相关。在本研究中，研究了TNF-α对TROP-2表达调控的影响及其在结肠癌细胞迁移和侵袭中的作用。使用蛋白质印迹分析评估在不同浓度TNF-α培养的HCT-116人结肠癌细胞中TROP-2蛋白水平。分别使用伤口愈合试验和Transwell试验评估细胞迁移和侵袭潜能的变化。然后，通过使用小干扰RNA（siRNA）下调细胞中的TROP-2表达，并分别通过逆转录定量聚合酶链反应和蛋白质印迹在mRNA和蛋白质水平确认TROP-2敲低。还评估了用TROP-2 siRNA转染的细胞的迁移和侵袭潜能。使用蛋白质印迹分析检测在用TNF-α处理的细胞中几种丝裂原活化蛋白激酶蛋白的水平，即p38、c-Jun氨基末端激酶（JNK）和细胞外信号调节激酶（ERK）。结果表明，与未处理的对照相比，用较低浓度TNF-α处理的细胞中TROP-2蛋白水平升高，但用较高浓度TNF-α处理的细胞中TROP-2蛋白水平降低。在用20μg/l TNF-α处理的细胞中观察到最高TROP-2水平。用20μg/l TNF-α处理的细胞中迁移和侵袭增强。当通过siRNA使结肠癌细胞中的TROP-2沉默时，与对照细胞相比，迁移和侵袭显著降低。TNF-α刺激激活ERK1/2途径，但未显著影响p38和JNK磷酸化水平。用特异性ERK1/2抑制剂处理可抑制TNF-α诱导的TROP-2上调以及TNF-α诱导的结肠癌细胞迁移和侵袭。总之，本研究结果表明，低浓度TNF-α通过ERK1/2信号通路上调TROP-2，显著增强结肠癌细胞的迁移和侵袭。
