| Objective:To investigate the effects of TF/VIIa/PAR2aixs on colon cancer line SW620cell proliferation, migration and apoptosis; to analyze the expression of apoptosis regulatory proteins--cysteine protease-3(caspase-3). matrix metalloproteinase-9(MMP-9), the adhesion molecule CD44; and to further explore the effects of calcium signaling, MAPKs/NF-κB signal transduction pathways in TF/VIIa/PAR2axis caused cell proliferation and migration.Methods:(1) SW620cells were treated with VIIa (10nM) and PAR2-AP (100μM) for different times, then the cell proliferation and migratory capacities were detected by cell counting method and wound healing assay, respectively.(2) Real-time fluorescence quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to detect the expression of MMP-9and CD44molecule on SW620cells after PAR2-AP (100μM) and VIIa (10nM) stimulation, to analyze the effects of PAR2-AP andFVIIa on MMP-9and CD44expression.(3) SW620cells were treated with different stimulants including VIIa (10nM). PAR2-AP (100μM), PAR2antagonist (PAR2-aAP,100μM), anti-TF antibody (a-TF,10μg/ml). and MOPC-21(10μg/ml). the expression of caspase-3in above treated cells were detected by real-time fluorescence quantitative PCR and Western blotting, in order to evaluate whether the effects of VIIa were dependent on TF or PAR2.(4) SW620cells were pre-incubated with p38MAPK inhibitor (SB203580,10μM). ERK1/2inhibitor (U0126,10μM) and NF-κB inhibitor (PDTC,5μM) for30min, then stimulated by PAR2-AP (100μM) and VIIa (10nM). the expressions of caspase-3. MMP-9and CD44in treated cells was measured to analyze the inhibitory effects of SB203580, U0126and PDTC, in order to elucidate the signal transduction pathways of these molecules expression regulated by TF/VIIa/PAR2.(5) SW620cells loaded with fluo-4/AM were treated with VIIa and PAR2-AP, then the changes of intracellular calcium fluorescence intensity (FI) were detected with fluo-4/AM using confocal laser scanning microscopy. To evaluate the role of calcium signaling after the activation of TF/VIIa/PAR2axis.(6) SW620cells were pretreated with calcium inhibitors including EGTA (1mM) and Thapsigargin (TG,1μM). then observed①the effects of calcium inhibitors to p-ERK1/2protein with VIIa and PAR2-AP stimulation, and②the effects of calcium inhibitors to cell proliferation, cell cycle and cell cycle regulated by FVIIa or PAR2-AP. to clarity the role of calcium signaling after TF-VII/PAR axis activationResults:(1)VIIa(10nM) could significantly promote the growth and migration ability of SW620cells (p<0.05vs control), similar to the effect of PAR2-AP (100uM).(2) Both FVIIa (10nM) and PAR2-AP (100μM) could decrease caspase-3mRNA and protein levels, increase the expression of MMP-9and CD44molecules.(3) Both PAR2-aAP (100μM) and a-TF (10μg/ml) could significantly inhibit the effects of Vila on caspase-3expression, but isotype control antibody (MOPC-21.10μg/ml) could not.(4) The effects of Vila or PAR2-AP on caspase-3, MMP-9and CD44expression could be obviously blocked by the inhibitors of ERK1/2(U0126,10μM) and NF-κB (PDTC,5μM). The inhibitor of p38MAPK (SB203580,10μM) could intervene in the regulatory effects of Vila on MMP-9and CD44, but not on caspase-3expression in the cells.(5) Vila (10nM) and PAR2-AP (100μM) could rapidly increase [Ca2+]i in SW620cells, reached the peak phase, then followed by a delayed phase, While the control group has no changes.(6) Vila and PAR2-AP could induce ERK1/2activation and their effects could be inhibited by EGTA and TG.(7) Both VIIa (10nM) and PAR2-AP (100μM) could respectively increase the rate of S phase of SW620cells, decrease G0/G1phase rate, raise the ability of cell proliferation and migration potential, such effects of VIIa and PAR2-AP could be partially blocked by the inhibitor of EGTA and TG.Conclusions:(1) VIIa dependent on TF and PAR2activation promotes colon cancer cell lines SW620cells proliferation and migration.(2)FVIIa dependent on TF and PAR2activation reduces caspase-3expression, and increases MMP-9and CD44molecule expression.(3) VIIa dependents on TF and PAR2activation, via ERK1/2and NF-κB signaling pathways, reduces caspase-3expression, increases MMP-9and CD44molecule expression, and induces SW620cells proliferation and migration.(4) VIIa dependent on TF and PAR2activation, triggers calcium signaling, thereby activates down-stream ERK1/2molecules, and induces SW620cells proliferation and migration. (5) TF/VIIa/PAR2axis, via calcium signaling and MAPKs/NF-κB signaling transduction pathways, regulates the expression of some effector molecules and promotes SW620cells proliferation and migration. |
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