| Objective:The effect of paeoniflorin on the migration and invasion of human glioma cells was observed by culturing human glioma cell lines(U87 and U251)with paeoniflorin,and looking for the target and related molecular mechanism which were responsible for the biological behaviour changes of glioma cells.Methods:U87,U251 cells were cultured with different concentrations of paeoniflorin and the impact of paeoniflorin on cell proliferation was tested by CCK-8 assay.Wound scratch assay and Transwell assay were used to detect the abilities of migration and invasion respectively.After treatment with different concentrations of paeoniflorin,the expressions of migration and invasion related proteins(MMP-2,MMP-9),the mark proteins of mesenchymal cells(N-cadherin,snail,vimentin,zeb-1)and tight junction proteins(claudin-1,zo-1)in each group were measured by Western Blot.RT-qPCR and Western Blot were uncover to test the mRNA level and protein level of TGF-b in each group.U87 and U251 cells were infected with the lentiviral vector which carried the TGF-? gene.Cells which had the ability of stably expressing TGF-? for subsequent experiments were screened by puromycin,and the effect of infection was assessed by Western Blot.The applications of Wound scratch assay and Transwell assay were used for detecting whether over-expressed TGF-b can neutralize the influence of paeoniflorin on the biological behaviour of glioma cells,and the proteins which were mentioned before were exmined by Western Blot.Nude mice were transplanted with U87 cells.After tumor formation,these mice were randomly divided into two groups.One group was given PBS solution and the other group was given different concentrations of paeoniflorin.The size of the transplanted tumor was observed every 4 days.After 36 days the nude mice were sacrificed and the tumors removed.The expression of related proteins in nude mice were detected by Western Blot Results:CCK-8 assay revealed that 15 mM concentration of paeoniflorin could inhibt the proliferation of U87 and U251 cells effectively,however the effect of 5mM and 10 mM paeoniflorin on the proliferation of U87 and U251 cells was not statistically significant.U87 cand U251 cells were both culture with 0 mM,5mM,10 mM paeoniflorin respectively.Wound scratch assay and Transwell assay showed that paeoniflorin could suppress cell migration and invasion,and the degree of suppression was proportional to the drug concentration.The results of Western Blot indicated that proportional could down-regulate the expressions of MMP-2,MMP-9,N-cadherin,snail,vimentin,zeb-1,and up-regulate claudin-1 and zo-1 protein expression.Meanwhile,the mRNA level and protein level of TGF-b were both decreased in groups with proportional.The results of Wound scratch assay and Transwell assay demonstrated that over-expression of TGF-b could inhance cell migration and invasion abilities.Western Blot showed that in cells which translated more TGF-b,the expression levels of MMP-2,MMP-9,N-cadherin,snail,vimentin and zeb-1 were increased and the expressions of claudin-1 and zo-1 were inhibited.In addition,we found that paeoniflorin restrained the growth of glioma in vivo.Western Blot revealed that the levels of claudin-1 and zo-1 in paeoniflorin group were higher than PBS group and the expressions of MMP-2,MMP-9,N-cadherin,snail,vimentin,zeb-1 and TGF-b in paeoniflorin group were less than PBS group.Conclusions:Paeoniflorin can inhibit the abilities of migration and invasion in U87 and U251 cells.The effect of paeoniflorin on the expression of TGF-? in glioma cells affects the expression of various effector proteins in the downstream.These effector proteins prevent glioma cells epithelial-mesenchymal transition(EMT),and thus inhibit the migration and invasion of glioma cells.In vivo,paeoniflorin suppresses glioma growth in the same molecular mechanism. |
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