Mechanism of siRNA-mediated silencing of DJ-1 via the PTEN/Akt pathway on human gastric cancer cells
Objective To investigate the effect of siRNA silencing DJ-1 gene on human gastric cancer MGC803 cells in vitro and in vivo.Methods MGC803 cells silenced DJ-1 gene were constructed.The effects of silencing DJ-1 gene on proliferation,clonal formation,migration and invasion of MGC803 cells were determined by methyl thiazolyl tetrazolium(MTT),plate cloning,cell scratch and invasion assay.The expressions of DJ-1,PTEN,Akt,p-Akt,Snail,Vimentin,E-cadherin,matrix metalloproteninases-9(MMP-9)and tissue inhibitor-3 of matrix metalloproteinases(TIMP3)were de-tected by real-time quantitative fluorescent PCR(qRT-PCR)and western blot.The effect of DJ-1 silencing on the mor-phology of MGC803 cells was observed by contrast microscope.The effect of DJ-1 silencing on MGC803 cell transplanted tumor was detected in nude mice.Results Four groups of si-DJ-1 and si-NC plasmid were transfected into MGC803 cells respectively.The expression of DJ-1 mRNA and protein in si-DJ-1A group was significantly down-regulated(all P＜0.001).si-DJ-1A was subsequently selected as MGC803 cells with stable DJ-1 gene silence.MTT showed that after 24,48 and 72 h,the proliferative activity of si-DJ-1 group was lower than that of MGC803 control group and si-NC group,respectively,P＜0.001.The clone formation experiment showed that the number of clone formation in si-DJ-1 group was lower than that in control group and si-NC group(P＜0.001).The results of scratch test showed that the migration dis-tance of si-DJ-1 group[(199.21+14.74)μm]was shorter than that of control group[(302.62+17.70)μm]and si-NC group[(304.49+13.55)μm],P＜0.001.Invasion experiment showed that the number of invaded cells in si-DJ-1 group(44.80±5.10)was lower than that in control group(83.80±5.80)and si-NC group(82.80±5.80),P＜0.001.Com-pared with the control group and the si-NC group,the DJ-1 mRNA(F=18.350,P=0.003),Snail mRNA(F=26.320,P＜0.001),Vimentin mRNA(F=44.379,P＜0.001)and MMP-9 mRNA(F=57.450,P＜0.001)were down-regula-ted,while E-cadherin mRNA(F=94.529,P＜0.001)and TIMP3 mRNA(F=16.480,P=0.004)were up-regulated.In si-DJ-1 group,DJ-1 protein(F=6.393,P=0.004),p-Akt(F=12.980,P=0.007),Snail(F=381.000,P＜0.001),Vimentin(F=99.750,P＜0.001)and MMP-9(F=126.800,P＜0.001)were down-regulated,while PTEN(F=36.880,P＜0.001),E-cadherin(F=181.000,P＜0.001)and TIMP3(F=217.800,P＜0.001)were up-regula-ted.Phase contrast microscopy showed that the fibroblast-like long spindle cell tumor stem cells in si-DJ-1 group de-creased significantly,the number of round and oval cells increased,and the atypia decreased.In vivo experiment showed that the growth rate of transplanted tumor in si-DJ-1 group was slower than that in control group,and the mass of trans-planted tumor in si-DJ-1 group[(0.51±0.05)g]was lower than that in control group[(0.90±0.16)g],t=5.273,P＜0.001.Compared with the control group,the positive expressions of DJ-1,Ki-67,Vimentin and CD-34 in si-DJ-1 group were decreased,while the expressions of PTEN and E-cadherin were increased.Conclusion DJ-1 silencing can in-hibit proliferation,migration,invasion and EMT of MGC803 cells through PTEN/Akt pathway in vitro and in vivo.
human gastric cancer MGC803 cellsDJ-1 silentPTEN/Akt pathwayproliferationmigrationinvasionepi-thelial-mesenchymal transition
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