肿瘤坏死因子α诱导蛋白8通过调控上皮-间充质转化影响前列腺癌细胞的侵袭与转移

Tumor necrosis factor alpha induced protein-8 affects the invasion and migration of prostate carcinoma cells by regulating the epithelial-mesenchymal transition

目的探讨前列腺癌细胞内肿瘤坏死因子α诱导蛋白8(TNFAIP8)的表达变化对其侵袭与转移能力的影响,及在此过程中有无上皮-间充质转化(EMT)参与。方法设计合成靶向抑制TNFAIP8基因的短发卡RNA(shRNA)慢病毒质粒(shTNFAIP8),转染前列腺癌PC3细胞,分为shTNFAIP8组(转染TNFAIP8 shRNA),空载细胞组(转染对照shRNA)和阴性对照组(未转染),反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测3组TNFAIP8和EMT相关标志物E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)和紧密连接蛋白(ZO1) mRNA及蛋白表达水平,通过细胞划痕和Transwell小室侵袭实验检验3组细胞侵袭与转移能力。组间比较采用t检验。结果 shTNFAIP8组Vimentin和N-cadherin mRNA及蛋白表达量(0.51±0.17、0.32±0.11、0.29±0.04、0.25±0.05)明显低于阴性对照组(1.00±0.32、1.00±0.23、0.63±0.10、0.65±0.07,t= 3.024、5.964、7.059、10.401,P<0.05)及空载细胞组(1.00±0.26、1.00±0.26、0.61±0.13、0.63±0.08,t=3.527、5.386、5.261、9.007,P<0.05),而E-cadherin和ZO1 mRNA及蛋白表达量(4.21±1.12、2.70±0.87、0.49±0.07、0.52±0.08)明显高于阴性对照组(1.00±0.14、1.00±0.26、0.31±0.06、0.39±0.05,t= 6.359、4.186、4.679、3.081,P<0.05)及空载细胞组(1.00±0.21、1.00±0.24、0.27±0.05、0.39±0.07,t=6.299、4.212、5.719、2.735,P<0.05),差异有统计学意义。shTNFAIP8组24 h后的划痕愈合百分比[(0.72±0.12)%]高于阴性对照组[(0.52±0.11)%]及空载细胞组[(0.53±0.12)%],差异有统计学意义(t=2.747、2.372,P<0.05)。shTNFAIP8组穿膜细胞数[(52±17)个]少于阴性对照组[(141±34)个]及空载细胞组[(150±45)个],差异有统计学意义(t=5.235、4.555,P<0.05)。结论下调表达TNFAIP8可通过调控EMT抑制前列腺癌细胞的侵袭与转移。

Objective To investigate the effect of tumor necrosis factor alpha induced protein-8(TNFAIP8)on invasion and migration of prostate cancer cells,and assess whether epithelial-mesenchymal transition(EMT)is involved in this process.Methods Short hairpin RNA(shRNA)lentiviral vector targeting TNFAIP8(shTNFAIP8)was designed and synthesized and transfected into PC3 cells.It was divided into 3 groups:shTNFAIP8 group(transfected with TNFAIP8 shRNA),blank load transfection group(transfected with control shRNA)and negative control group(untransfected).TNFAIP8 and molecular markers associated with EMT(E-cadherin,ZO1,Vimentin,N-cadherin)were detected by reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting.The cell invasion and metastasis abilities of each group were tested by cell scratch test and Transwell invasion test.The t test was used for analysis between the two groups.Results The mRNA and protein expression of Vimentin and N-cadherin in shTNFAIP8 group(0.51±0.17,0.32±0.11,0.29±0.04,0.25±0.05)were significantly lower than those in negative control group(1.00±0.32,1.00±0.23,0.63±0.10,0.65±0.07,t=3.024,5.964,7.059,10.401,P<0.05)and blank load transfection group(1.00±0.26,1.00±0.26,0.61±0.13,0.63±0.08,t=3.527,5.386,5.261,9.007,P<0.05),and the mRNA and protein expression levels of E-cadherin and ZO1(4.21±1.12,2.70±0.87,0.49±0.07,0.52±0.08)were significantly higher than those of negative control group(1.00±0.14,1.00±0.26,0.31±0.06,0.39±0.05,t=6.359,4.186,4.679,3.081,P<0.05)and blank load transfection group(1.00±0.21,1.00±0.24,0.27±0.05,0.39±0.07,t=6.299,4.212,5.719,2.735,P<0.05).The percentage of wound closure of shTNFAIP8 group[(0.72±0.12)%]after 24 h was significantly higher than that of negative control group[(0.52±0.11)%]and blank load transfection group[(0.53±0.12%,t=2.747,2.372,P<0.05].The number of penetrating cells in shTNFAIP8 group(52±17)was significantly lower than that in negative control group(141±34)and blank load transfection group(150±45,t=5.235,4.555,P<0.05).Conclusion Down-regulation of TNFAIP8 can inhibit the invasion and migration of prostate cancer cells by regulating EMT.