siRNA沉默DJ-1通过PTEN/Akt通路对人胃癌细胞影响机制

Mechanism of siRNA-mediated silencing of DJ-1 via the PTEN/Akt pathway on human gastric cancer cells

目的探讨siRNA沉默DJ-1基因体内外对人胃癌MGC803细胞的影响机制。方法构建沉默DJ-1基因MGC803细胞;四甲基偶氮唑蓝(MTT)比色法、平板克隆、细胞划痕和侵袭实验检测沉默DJ-1基因对MGC803细胞增殖、克隆形成、迁移与侵袭的影响;实时荧光定量PCR(qRT-PCR)和蛋白质印迹法检测DJ-1、PTEN、Akt、p-Akt、Snail、Vimentin、E-cadherin、基质金属蛋白酶9(MMP-9)与基质金属蛋白酶组织抑制因子3(TIMP3)的表达;相差显微镜观察DJ-1沉默对MGC803细胞形态学的影响;裸鼠实验检测DJ-1沉默对MGC803细胞移植瘤的影响。结果分别用4组si-DJ-1及si-NC质粒转染MGC803细胞,其中si-DJ-1A组DJ-1 mRNA与蛋白表达下调较为显著(P<0.001),则后续选用si-DJ-1A作为稳定沉默DJ-1基因MGC803细胞。MTT法检测结果显示,24、48和72 h后,si-DJ-1组增殖活性分别较MGC803对照组和si-NC组降低,均P<0.001。克隆形成实验显示,si-DJ-1组克隆形成数较对照组与si-NC组减少,P<0.001。划痕实验结果显示,si-DJ-1组迁移距离[(199.21±14.74)μm]较对照组[(302.62±17.70)μm]和si-NC组[(304.49±13.55)μm]缩短,P<0.001。侵袭实验显示,si-DJ-1组侵袭细胞数[(44.80±5.10)个]较对照组[(83.80±5.80)个]和si-NC组[(82.80±5.80)个]减少,P<0.001。与对照组和si-NC组相比,si-DJ-1组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)与MMP-9 mRNA(F=57.450,P<0.001)下调,而E-cadherin mRNA(F=94.529,P<0.001)与TIMP3 mRNA(F=16.480,P=0.004)上调;si-DJ-1组DJ-1(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)与MMP-9(F=126.800,P<0.001)蛋白下调,而PTEN(F=36.880,P<0.001)、E-cadherin(F=181.000,P<0.001)与TIMP3(F=217.800,P<0.001)上调。相差显微镜显示,si-DJ-1组纤维母细胞样长梭形细胞肿瘤干细胞减少,圆形与椭圆形细胞增多,异型性下降。体内实验表明,si-DJ-1组移植瘤生长速度较对照组减慢,si-DJ-1组移植瘤质量[(0.51±0.05)g]较对照组[(0.90±0.16)g]减小,t=5.273,P<0.001。si-DJ-1组较对照组DJ-1、Ki-67、Vimentin和CD-34阳性表达降低,而PTEN和E-cadherin表达增强。结论DJ-1沉默可通过PTEN/Akt通路体内外抑制MGC803细胞增殖、迁移、侵袭与上皮细胞-间充质转化。

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 detected by real-time quantitative fluorescent PCR(qRT-PCR)and western blot.The effect of DJ-1 silencing on the morphology 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 distance 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.Compared 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-regulated,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-regulated.Phase contrast microscopy showed that the fibroblast-like long spindle cell tumor stem cells in si-DJ-1 group decreased 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 transplanted 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 inhibit proliferation,migration,invasion and EMT of MGC803 cells through PTEN/Akt pathway in vitro and in vivo.