DADS Inhibit The EMT And Invasion In MGC803 Cells By Down Regulation Of DJ-1

Objective: The main objective of this paper is to investigate the inhibitory effect of DADS on the biological behavior of tumor cells and verify the effect of DJ-1 overexpression on the inhibition of EMT,migration and invasion in MGC803 cells by DADS,and to elucidate the role of DJ-1 in gastric cancer.It is concluded that DJ-1 is one of the targets of DADS to inhibit the migration and invasion of human gastric cancer cells and provide a theoretical basis for molecular therapy of clinical gastric cancer.Methods: Construction of DJ-1 overexpression plasmid for DJ-1 gene was transfected into gastric cancer MGC803 cell line by liposome.After8 weeks of screening by G418,the stable overexpression of DJ-1/MGC803 cells was established.The expression of DJ-1 protein and mRNA in MGC803 cells was detected by Western blot and qRT-PCR.The transfection efficiency of DJ-1 was verified.The relative biological behaviors of MGC803 cell lines were compared before and after treatment with DADS,and the effects of overexpression of DJ-1 and DADS on the biological behavior of MGC803 cells were verified.The changes in shape of MGC803 cells were observed by phase contrastmicroscope.MTT and plate clone were used to detect the inhibitory effect of DADS on the proliferation of MGC803 cells.Cell migration assay,scratch test and cell invasion assay were used to detect the migration and invasion of MGC803 cells.The expressions of DJ-1,PTEN,AKT,p-AKT,Vimentin,E-cadherin,MMP-9,TIMP-3,Snail mRNA and protein in MGC803 cells were detected by qRT-PCR and Western blots.The effect of DADS on the tumorigenesis of MGC803 cells in nude mice was observed by nude mice.Results:1.The MGC803 cell line with stable overexpression of DJ-1 was successfully constructedThe expression vector of DJ-1 and empty vector were transfected into MGC803 cells.The expression of DJ-1 in MGC803 cells was significantly increased by Western blot and q RT-PCR.Then,the transfected cells were screened with G418,and the stable overexpressing of DJ-1 in MGC803 cell line was established.2.The morphological changes of MGC803 cells were observed by phase contrast microscopePhase contrast microscopy showed that MGC803 cells showed long spindle shape,atypia significantly.DJ-1 overexpressing MGC803 cells was more pronounced than the former.After 24 hours of treatment with DADS,MGC803 cells had decreased spindle shape,increased oval shapeand decreased atypia.DADS-treated DJ-1 overexpressed cells were partially rounded and spindle cells were reduced.3.Effects of DADS and DJ-1 on the proliferation of MGC803 cellsThe number of clones formed by MGC803 cells and DJ-1 high expression group was 27.7±1.15 and 34.7±1.53 respectively after DADS treatment.Compared with the untreated MGC803 and DJ-1overexpression group clones formed 45.3±2.08,86.3±1.53 significantly decreased(P<0.05).The results showed that DJ-1 overexpression could promote the proliferation of MGC803 cells,while DADS could inhibit the proliferation of MGC803 cells.Migration experiments show that there was no significant difference in the number of migrating cells between the MGC803 group and the empty vector group at 116±1.22 and 127±1.58(P>0.05).The number of migrating cells was significantly higher than that of MGC803 and empty vector group(P<0.05).After 24 hours of DADS treatment,the migration of MGC803 group and empty vector group was 124±1.58,which was significantly lower than that of the untreated group(P<0.05).Scanning experiments showed that the migration distance of DJ-1 group was significantly higher than that of MGC803 group 13.77±4.33 and 14.98±3.17(P<0.05).The migration distance of DJ-1 group was significantly lower than that of the control group(P<0.05)and the distance of 9.61±1.82 in MGC803 group and 9.74±0.69 in empty vector group wassignificantly lower than that before treatment(P<0.05).The results showed that the DJ-1 overexpression could promote the migration ability of MGC803 cells and attenuate the inhibitory effect of DADS on the migration of MGC803 cells(P<0.05).Invasion experiments show that the number of transmembrane cells in MGC803 group and empty vector group was 67.4±1.14 and 62.8±2.77,there was no significant difference(P>0.05).The overexpression of DJ-1group was 86.6±1.52 significantly higher than that of MGC803 and empty vector group(P<0.05).After treatment with DADS,the transmembrane cells of MGC803 group,empty vector group and DJ-1overexpression group were 54.2±1.92,50.2±1.64 and 55.4±1.52 respectively,which were significantly reduced than those before treatment(P<0.05).Indicating that over expression of DJ-1 can promote the invasion of MGC803 cells,while DADS can inhibit the invasion of MGC803 cells.5.The effect of DADS on the expression of DJ-1/PTEN/p-AKT in MGC803 cellsQRT-PCR results show the expression of DJ-1 mRNA in the empty vector group was not significantly different from that in the MGC803 cell group.Compared with MGC803 cells,the expression of DJ-1 mRNA was significantly up-regulated and the expression of PTEN mRNA was down-regulated in DJ-1 overexpression cells(P<0.05).After treatmentwith DADS,the expression of DJ-1 mRNA was down-regulated and the expression of PTEN mRNA was up-regulated compared with untreated group(P<0.05).Western blot showed There was no significant difference in the expression level of DJ-1 protein between MGC803 group and empty vector group(P>0.05).The expression of DJ-1 was significantly up-regulated in DJ-1 overexpression cells(P<0.05).The expression of DJ-1 and p-Akt in MGC803 cells,empty vector group and DJ-1overexpression cells was significantly lower than that in untreated group(P<0.05).The expression level of AKT protein in the cells before and after treatment did not change significantly.Indicating that DADS can downregulate DJ-1,p-Akt protein and up-regulate PTEN.The results were consistent with the results of qRT-PCR.Cell immunofluorescence showed that DJ-1 fluorescence was significantly enhanced in DJ-1 cells and PTEN was significantly decreased.After treatment with DADS,the fluorescence intensity of DJ-1was decreased and the PTEN fluorescence was significantly increased.The results were consistent with q RT-PCR and Western Blot.6.Effects of DADS on EMT-related markers in MGC803 cells.QRT-PCR results show that compared with MGC803 cells,the expression of Vimentin,MMP-9 and Snail mRNA was significantly up-regulated and the expression of E-cadherin mRNA wasdown-regulated in DJ-1 overexpression cells(P<0.05).The expression of Vimentin,MMP-9 and Snail mRNA was down-regulated and the expression of E-cadherin mRNA was increased in DADS group(P<0.05).Indicating that DADS down-regulation of DJ-1 can inhibit EMT of MGC803 cells.Western blot showed that the expression of Vimentin,MMP-9 and Snail protein was significantly up-regulated and the expression of E-cadherin was down-regulated in DJ-1 overexpression cells(P<0.05)compared with that of MGC803 cells.The expression of Vimentin,MMP-9 and Snail protein in DADS group was lower than that in untreated group,while the expression of E-cadherin protein was up-regulated(P<0.05).Immunofluorescence showed that the expression of Vimentin and MMP-9 protein in MG-C803 cells was significantly higher than that in MGC803 cells and E-cadherin fluorescence.The fluorescence intensity of Vimentin and MMP-9 decreased significantly after treatment with DADS,and the fluorescence of E-cadherin was significantly increased.The results were consistent with qRT-PCR and Western Blot.7.Effects of DADS and overexression of DJ-1 on tumorigenic ability of MGC803 cellsNude mice showed tumor formation,the tumor size and weight of the transplanted tumor in the DJ-1 overexpression cell group weresignificantly increased than MGC803 cell group(P<0.05).The volume and weight of the transplanted tumor of DJ-1 overexpression group and MGC803 cell group were significantly decreased after DADS treatment(P<0.05).Indicating that DJ-1 overexpression can promote the tumorigenic ability of MGC803 cells in nude mice,and DADS can significantly inhibit the tumorigenic ability of nude mice.Conclusion:1.Overexpression of DJ-1 could promote the proliferation of MGC 803 cells,EMT and migration invasion and tumorigenic ability.2.DADS can inhibit the proliferation,EMT,migration and tumorigenesis of MGC803 cells by blocking the DJ-1/PTEN/AKT pathway.