Jia Yitao, Zhang Suqiao, Miao Lingling, Wang Jingbao, Jin Zujian, Gu Bin, Duan Zhihui, Zhao Zhaolong, Ma Shunmao, Zhang Wenjin, Li Zhongxin
Department of Oncology, Hebei General Hospital, Shijiazhuang, Hebei 050051, P.R. China.
Second Department of Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China.
Oncol Rep. 2015 Jun;33(6):2681-8. doi: 10.3892/or.2015.3897. Epub 2015 Apr 3.
The aim of the present study was to examine the role of protease-activated receptor-1 (PAR1)-stimulated platelet activation in the epithelial-mesenchymal transition (EMT) and migration of colon cancer cells, and to identify the underlying mechanisms. TFLLR-NH2, a PAR1 agonist, was used to activate platelets and the platelet supernatants were used to treat the SW620 colon cancer cell line. Expression of E-cadherin and vimentin on SW620 cells was detected by immunofluorescence and western blotting, and the level of the transforming growth factor β1 (TGF-β1) was measured using ELISA following the activation of platelets by TFLLR-NH2. miR-200b expression was detected using quantitative PCR in SW620 cells. In order to investigate the chemotactic ability of the SW620 cells, the expression of CXC chemokine receptor type 4 (CXCR4) was measured by flow cytometry. Transwell migration assays were performed following exposure of the cells to the supernatant of PAR1-activated platelets. SW620 cells cultured in the supernatant of TFLLR-NH2-activated platelets upregulated E-cadherin expression and downregulated the vimentin expression. In the in vitro platelet culture system, a TFLLR-NH2 dose-dependent increase of secreted TGF-β1 was detected in the supernatant. The activation of PAR1 on the platelets led to the inhibition of miR-200b expression in the SW620 cells that were cultured in platelet-conditioned media. The number of SW620 cells that penetrated through the Transwell membrane increased with the dose of TFLLR-NH2 used to treat the platelets. The percentage of CXCR4-positive SW620 cells was significantly higher when they were exposed to the supernatant of platelets cultured for 24 h with PAR1 agonist than when cultured in non-conditioned media (40.89 ± 6.74 vs. 3.47 ± 1.40%, P < 0.01). Platelet activation with a PAR1 agonist triggered TGF-β secretion, which induced EMT of SW620 human colon cancer cells via the downregulation of miR-200b expression, and activated platelets had a chemotactic effect on colon cancer cells mediated by the upregulation of CXCR4 on the cell surface.
本研究的目的是探讨蛋白酶激活受体-1（PAR1）刺激的血小板活化在结肠癌细胞上皮-间质转化（EMT）和迁移中的作用，并确定其潜在机制。使用PAR1激动剂TFLLR-NH2激活血小板，并用血小板上清液处理SW620结肠癌细胞系。通过免疫荧光和蛋白质印迹法检测SW620细胞上E-钙黏蛋白和波形蛋白的表达，并在TFLLR-NH2激活血小板后使用酶联免疫吸附测定法（ELISA）测量转化生长因子β1（TGF-β1）的水平。使用定量PCR检测SW620细胞中miR-200b的表达。为了研究SW620细胞的趋化能力，通过流式细胞术测量CXC趋化因子受体4（CXCR4）的表达。在细胞暴露于PAR1激活的血小板上清液后进行Transwell迁移试验。在TFLLR-NH2激活的血小板上清液中培养的SW620细胞上调了E-钙黏蛋白的表达并下调了波形蛋白的表达。在体外血小板培养系统中，检测到上清液中TFLLR-NH2剂量依赖性增加的分泌型TGF-β1。血小板上PAR1的激活导致在血小板条件培养基中培养的SW620细胞中miR-200b表达受到抑制。穿透Transwell膜的SW620细胞数量随着用于处理血小板的TFLLR-NH2剂量的增加而增加。当SW620细胞暴露于用PAR1激动剂培养24小时的血小板上清液时，CXCR4阳性SW620细胞的百分比显著高于在非条件培养基中培养时（40.89±6.74对3.47±1.40%，P<0.01）。用PAR1激动剂激活血小板触发TGF-β分泌，其通过下调miR-200b表达诱导SW620人结肠癌细胞的EMT，并且活化的血小板对细胞表面CXCR4上调介导的结肠癌细胞具有趋化作用。
