The Effects Of Upregulating MiR-7 For The EMT And Invasion In SGC-7901 Inhibited By DADS

Objective: This study aims to clarify the fuction of DADS regulating the mi R-7,providing theoretical and experimental basis for the development of gastric cancer.And elucidate the molecular mechanism that the migration and invasion of gastric cancer cells could be inhibited through EGFR-Rac1-Pak1 pathway regulated by DADS upregulating the mi R-7,providing a new target for developing the new anti-gastric cancer drugs.Methods: mi R-7 silenced lentivirus infected the human gastric cancer SGC7901,and then stable mi R-7 silenced SGC7901 cell line was established.Morphological changes of SGC7901 cells were observed using light microscope after treated with DADS.Treated with 30mg/L DADS,the ability of mi R-7 silenced and DADS on proliferation,migration and invasion of SGC7901 cells were assessed by MTT assay,scratch woud healing assay and Transwell invasion assay.Then,Immunoflurescence assay,Western Blot and q RT-PCR were used to determine the expression of EGFR?RAC-1?PAK-1?E-cadhein and Vimentin in SGC7901 cells.Result: 1.Establish stable mi R-7 silenced SGC-7901 cells successfully.mi R-7 silenced lentivirus and negative control lentivirus were used to infect SGC-7901 cells.q RT-PCR showed that the mi R-7 in infected group was significantly lower than that uninfected group(P<0.05),which suggested that stable mi R-7 silenced SGC-7901 cells was established successfully.2.Morphological changes of SGC7901 cells dealing with DADS.The observation of phase contrast microscope showed that the cells in antago-mi R-7 group were significantly more irregular than the SGC7901 group,and the shape of the cells was large.After treated with DADS,the cell atypia of each group was decreased and the shape became round.The results indicated that DADS could inhibit EMT,and silenced mi R-7 could promote EMT.3.Effects of DADS on the proliferation of mi R-7 silenced SGC-7901 cells.MTT results showed that with the extension of time,living cells cells were increased.The OD value of SGC7901,vector,mi R-7-antago,SGC7901+DADS,vector+DADS,mi R-7-antago+DADS groups in 0 hours were 0.217 + 0.008,0.201 + 0.017,0.219 + 0.017,0.202 + 0.024,0.220 + 0.019,0.207 + 0.016,while 72 hours being respectively 1.084 +0.014,1.084 + 0.010,1.304 + 0.011,0.698 + 0.008,0.702 + 0.010,0.890 + 0.015.The results indicated that silenced mi R-7 could promote the proliferation of SGC-7901 cells,DADS inhibited the proliferation of SGC-7901 cells,and DADS could inhibit the proliferation of mi R-7.4.Effects of DADS on migration and invasion of mi R-7 silenced SGC-7901 cells.The scratch test showed that migration rate(%)of SGC7901,vector,mi R-7-antago,SGC7901+DADS,vector +DADS,mi R-7-antago +DADS were 16.8 + 1.2,15.7 + 0.7,33.3 + 1.6,8.4 + 0.9,5.2 + 0.8,14.9 + 1.7.Transwell invasion results showed that number of transmembrane cells SGC7901,SGC7901+DADS,vector,vector,+DADS,mi R-7-antago,mi R-7-antago and +DADS were 81.8 + 5.8,50.6 + 5.2,78.8 + 5.8,49.6 + 8.4,138.8 + 5.1,98.8 + 2.6.Results show that mi R-7 silenced enhanced the ability of migration and invasion of SGC-7901 cells(P<0.05),and after treated with DADS,the ability of migration and invasion of SGC-7901 cells decreased(P<0.05),and DADS could reduce the ablity of silenced mi R-7 promoting tumor migration and invasion.5.Effects of DADS on the expression of mi R-7,EGFR,RAC-1 and PAK-1 in different groups q RT-PCR showed that the mi R-7 in each group of SGC-7901 cells after treatment with DADS was significantly higher than that in untreated group(P<0.05).The results of q RT-PCR and Western Blot showed that the expression of EGFR,RAC-1 and PAK-1 were significantly up-regulated in mi R-7 silencing group(P<0.05).Compared with untreated groups,the expression of EGFR,RAC-1 and PAK-1 was significantly lower in treated groups(P<0.05).Immunofluorescence showed that the fluorescence intensity of EGFR,RAC-1 and PAK-1 increased in mi R-7 silence group,and the fluorescence intensity of EGFR,RAC-1 and PAK-1 decreased after being treated with DADS,and the results were consistent with the results of q RT-PCR and Western Blot.6.Effect of DADS on EMT expression in SGC-7901 cells of each group The result of Western Blot showed that the expression of Vimentin protein in mi R-7 silenced group was higher than that in control group and blank group,but the expression of E-cadherin was decreased(P<0.05).After being treated with DADS,the expression of Vimentin protein was decreased,and the expression of E-cadherin was increased(P<0.05),which showed silenced mi R-7 up regulated the expression of Vimentin,and down-regulated E-cadherin;DADS down-regulated the expression of Vimentin,and can increase the expression of E-cadherin(P< 0.05).Immunofluorescence showed that fluorescence intensity Vimentin in mi R-7 silenced group was increased,and the fluorescence intensity of E-caherin was decreased.After treated with DADS,fluorescence intensity Vimentin was decreased,and the fluorescence intensity of E-caherin was increased.The results is consistent to q RT-PCR and Western Blot.Conclusion: 1.DADS can inhibit the migration,invasion and EMT of SGC-7901 cell by up-regulating the mi R-7 that regulated EGFR/RAC1/PAK1 pathway.2.mi R-7 silenced enhance the ability of proliferation,migration and invasion of SGC7901 and can decrease the capacity of DADS inhibiting the proliferation,migration and invasion of SGC7901 cells.