The Role Of Platelet Protease Activated Receptor-1Activation Induce Colon Cancer Cells SW620Cell Epithelial-Mesenchymal Transition And Its Mechanism

Colorectal cancer is a kind of common human malignant carcinomas.Lymph nodes and distant metastases result in the death of cancer patients.Thefirst step of metastases is EMT(epithelial mesenchymal transition, EMT). Thecytokines and the transcription factors that inducing EMT include Snail, Twist,TGF-β, Tiam1ect. TGF-β is one of the most important factors,closelyassociated with metastasis.In recent years, the relationship of platelets and tumor progressionattracts more and more doctors’s attention. The activation of platelet PAR1canactivate alpha-granules. It makes an important role of tumor progression.Butit is unclear whether platelet PAR1activation could induce EMT.Objective: To investigate the mechanism of the activation of PAR1induce SW620cell EMT.Methods：1SW620is treated with a concentration gradient (0μM,1μM,3μM,5μM,7μM,9μM,10μM) of platelet PAR1agonist (TFLLR-NH2). MTT was usedto detect each group to compare the effect of proliferation. Apply cytometry todetect the expression of CD62P in platelet which was treated with theconcentration gradien (0μM,1μM,3μM,5μM,7μM,9μM,10μM) ofplatelet PAR1agonist (TFLLR-NH2).2Experimental groups:①Unactivated platelet suspension100μL+SW620cells (experimental control group)②activated platelet suspension100μL+SW620cells (experimental control group)③activated plateletsupernatant100μL+SW620cells (experimental group)④TGF-β12μL+SW620cells (positive control group)⑤TFLLR-NH20.04μL+SW620cells(pure agonist group)⑥medium100μL of+SW620cells (negative control group). After24h in the incubator, Confocal immunofluorescent was used todetect the expression of the E-cadherin and Vimentin protein in SW620cellwhich were stained by PathScan EMT Duplex IF Kit.3Experimental groups:①Unactivated platelet suspension100μL+SW620cells (experimental control group)②activated platelet suspension100μL+SW620cells (experimental control group)③activated plateletsupernatant100μL+SW620cells (experimental group)④TGF-β12μL+SW620cells (positive control group)⑤TFLLR-NH20.04μL+SW620cells(pure agonist group)⑥medium100μL of+SW620cells (negative controlgroup). After24h in the incubator,western blotting was used to detect theexpression of the E-cadherin and Vimentin protein in SW620cell.4ELISA was used to detect the releasate of TGF-β1in plateletsupernatant after activated by the concentration gradient (0μM,1μM,3μM,5μM, and7μM,9μM,10μM) of PAR1agonist (TFLLR-NH2).Results:1MTT experiment result: the OD value of the concentration gradientagonist group was no statistically significant difference (P>0.05). Flowcytometry result:the expession of CD62P in platelet which were treated withthe concentration gradient（%）(1μM3μM5μM7μM,9μM) were（43.52±1.5）%，（64.22±3.60）%,（54.46±1.82）%,（53.15±2.7）%,（54.63±3.66）%VS（14.35±0.86）%respectively. Compared with the controlgroup, the expession of CD62P in the concentration gradient groups was allhigher than that of the control group (P <0.05).2Immunofluorescence experiment result: Compared with the negativecontrol group, the expression of E-cadherin decreased while the expressionof Vimentin increased in those treatment groups. Only，the expression ofE-cadherin and the Vimentin of agonist group was similar to that of thenegative control group.3Western blot experiment result: at24h, Compared with the negativecontrol group, the expression of E-cadherin decreased while the expression ofVimentin increased in those treatment groups.the efficacy of PAR1activated platelet suspension group was higher than that of PAR1unactivated plateletsuspension group and PAR1activated platelet supernatant group. But,theexpression of Smad4in each group had no statistically significant difference.4ELISA experiment: The OD values of the control group andconcentration （1uM,3uM，5uM，7uM，9uM）PAR1agonist groups under450nm which could represent the relative content of TGF-β1were0.095±0.003，0.133±0.005，0.322±0.007，0.497±0.012，0.571±0.012，0.592±0.021.Compared with the control group,the OD value of each agonistgroup was all higher than that of the control group(P<0.01). While nostatistical difference was found between the7uM group and the9uM group(P>0.1).Conclusions:1The low dose of PAR1agonist (1uM,3uM,5uM,7uM,9uM)don’taffect the proliferation of SW620, but could activate the CD62P of platelet.2The activation of platelet PAR1may through TGF-β1/Non-Smadpathyway induced EMT in SW620cell.3Alpha-granules could release TGF-β1after platelet PAR1activation;Moreover,with the increase of PAR1agonist, TGF-β1which released byplatelet showed ascendant trend.