Study On The Role And Mechanism Of Platelet On ATC Invasion And Metastasis

Objective:Our previous clinical study showed that the elevated peripheral blood platelet(PLT)counts may be an adverse prognostic factor of anaplastic thyroid carcinoma(ATC)patients,suggesting that platelet counts may involved ATC progression.Platelet has been reported closely related to the distant metastasis of many carcinomas,thus we suppose that platelet may participate in ATC invasion and migration process.This study aims to explore the role of platelet on ATC cells invasion and migration and clarify its mechanism.Methods:Platelets were isolated from healthy adult venous blood in vitro.Transwell assays were employed to analyze the effect of platelets on the abilities of migration and invasion of ATC cells.Animal experiments further verify the impact of platelets on the invasion and metastasis of anaplastic thyroid carcinoma.Transwell assays were employed to analyze the impact of cell supernatants after co-culture of ATC cells and platelets on the abilities of migration and invasion of ATC cells.Flow cytometry was used to detect platelet activation after the stimulation of thrombin.Human cytokine antibody array was employed to detect and filter significantly different cytokines among simple anaplastic thyroid carcinoma cells group,co-incubated with platelets group and simple activated platelets group.Transwell experiments were used to further verify the filitered significant differences cytokines whether promote the invasion and migration of ATC cells.The CCR1 and CCR5,the specific receptors for CCL3,the mRNA and protein of which detected through real-time PCR and western blot assays in ATC cell lines.Western blot was used to detect the knockdownefficiency of CCR1 and CCR5 in anaplastic thyroid carcinoma cells,which were down-regulated by siRNA respectively,then transwell assays were employed to assess the impact on the ability of CCL3 promoting the invasion and migration of anaplastic thyroid carcinoma cells.We use molecular cloning technique to construct the CCR5 eukaryotic overexpression vector:pMSCV-CCR5,which used to establish CCR5 stable overexpression cell lines.Real-time PCR and western blot were employed for examing the expression of CCR5 mRNA and protein in stable lines.Transwell assays were employed to assess the impact of overexpressing CCR5 on the ability of CCL3 promoting the invasion and migration of anaplastic thyroid carcinoma cells.Western blot was used to detect the associated signaling pathways proteins expression.Results:Vitro and vivo experiments confirmed that platelets can obviously promote the invasioness and metastasis of anaplastic thyroid carcinoma cells.Transwell experiments found that the supernatant of cells co-cultured with platelets significantly increased the invasioness and metastasis of ATC cells.Flow cytometry confirmed that thrombin can activate platelets in vitro.CCL3,a cytokine filitered by human cytokine antibody assay,which was confirmed to promote the invasion and metastasis of ATC cells.Transwell experiments and siRNA technique showed that CCL3 was involved in promoting ATC cells invasioness and metastasis through CCL3-CCR5 axis.CCR5 stable overexpression cell lines were sucessfully constructed.Transwell experiments confirmed that overexpressed CCR5 can increase the abilities of CCL3 promoting the invasion and migration of anaplastic thyroid carcinoma cells.Western blot showed that CCL3-CCR5 axis is involved in the process of platelets promoting the invasion and metastasis of anaplastic thyroid carcinoma by activating the NF-?B signaling pathway to up-regulate the expression of MMP-1 protein.Conclusion:Platelet can promote the invasion and metastasis of ATC obviously.CCL3 that secreted by activated platelets played a critical role by binding with its receptor CCR5,and further activated the NF-?B signaling pathway to up-regulate the expression of MMP-1 protein so as to participate in the process of platelets promoting the invasion and metastasis of ATC.This discovery could lead to a new development of prognostic or predictive tools as well as the identification of potential drug targets in ATC.