荷载核基质结合区结合蛋白1短发卡RNA的溶瘤腺病毒联合多西他赛对激素敏感前列腺癌LNcap细胞的影响

Effect of docetaxel combined with oncolytic adenovirus carrying sequence binding protein-1 short hairpin RNA on hormone-sensitive prostate cancer LNcap cells

目的观察荷载核基质结合区结合蛋白1(SATB1)短发卡RNA(shRNA)的溶瘤腺病毒(ZD55-SATB1)联合多西他赛(DTX)在体外对激素敏感前列腺癌LNcap细胞的影响,并探讨作用机制。方法将LNcap细胞(购自上海中国科学院细胞库)分为5组进行干预,分别为磷酸盐缓冲液组(PBS)、多西他赛化疗药物组(DTX为2μg/L)、病毒对照组[ZD55-EGFP为10感染复数(MOI)]、病毒治疗组(ZD55-SATB1为10 MOI)、病毒联合化疗药物组(ZD55-SATB1+DTX为5 MOI+1μg/L)。Transwell检测细胞迁移和侵袭能力变化;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测细胞凋亡;蛋白质印迹法(Western blot)检测SATB1、腺病毒早期区1A基因(E1A)、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)与基质金属蛋白酶-2(MMP-2)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3、Caspase-8与bcl-2的表达。采用t检验。结果Transwell迁移结果显示,ZD55-SATB1联合DTX组干预LNcap细胞24 h后的细胞穿膜数为(28.20±3.19)个,与ZD55-SATB1组[(39.60±4.98)个](t=4.309,P<0.01)、ZD55-EGFP组[(52.60±6.02)个](t=8.001,P<0.01)、DTX组[(65.50±5.12)个](t=13.710,P<0.01)、PBS组为(106.60±5.18)个(t=28.820,P<0.01)比较,病毒与化疗药物治疗组穿膜细胞数显著降低,细胞迁移能力显著降低,差异有统计学意义;Transwell侵袭实验结果:联合组穿透Matrigel胶进入小室下层的细胞数为(23.60±3.58)个,与ZD55-SATB1组[(32.40±5.08)个](t=3.088,P<0.05)、ZD55-EGFP组[(39.60±2.70)个](t=7.610,P<0.01)、DTX组[(44.60±4.62)个](t=7.815,P<0.01)、PBS组为(98.20±4.32)个(t=28.820,P<0.01)比较,细胞侵袭能力明显降低,差异有统计学意义。TUNEL结果显示,各治疗组细胞凋亡率均高于PBS对照组[(5.92±0.90)%],但联合组[(34.48±4.25)%]与ZD55-SATB1组[(28.12±2.50)%](t=2.582,P<0.05)、ZD55-EGFP组[(23.30±3.03)%](t=4.752,P<0.01)、DTX组[(22.35±2.44)%](t=4.956,P<0.01)比较,细胞凋亡更加明显,差异有统计学意义。Western blot结果显示,联合组内SATB1蛋白表达下调,E-cadherin表达上调,Vimentin和MMP-2表达下调,Caspase-8和Caspase-3表达上调,bcl-2表达下调更为显著。E1A基因在联合组内正常表达,说明联合使用DTX不影响腺病毒复制增殖。结论ZD55-SATB1联合DTX在体外可以有效抑制LNcap细胞的迁移和侵袭能力,并诱导肿瘤细胞凋亡,效果优于单独使用ZD55-SATB1或DTX,其机制可能是通过上调E-cadherin同时下调Vimentin和MMP-2的表达抑制迁移侵袭能力,抑制了上皮-间充质转化(EMT)进程,并通过上调Caspase-8和Caspase-3,下调bcl-2诱导肿瘤凋亡。

Objective To observe the effects of oncolytic adenovirus carrying special AT rich sequence binding protein-1(SATB1)short hairpin RNA(shRNA)combined with docetaxel on the migration and invasion of hormone-sensitive prostate cancer LNcap cells,and explore the action mechanism.Methods The prostate cancer LNcap cells were divided into 5 groups:phosphate buffer group(PBS),docetaxel group(DTX;2μg/L),viral control group[ZD55-enhanced green fluorescent protein(EGFP);10 multiplicity of infection(MOI)],virus treatment group(ZD55-SATB1;10 MOI),and virus combination DTX group(ZD55-SATB1+DTX;5 MOI+1μg/L).Transwell test was used to detect the change of cell migration and invasion ability.Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)test was used to detect the cell apoptosis in each group.The expression of SATB1,E1A,invasion-related proteins E-cadherin,Vimentin and matrix metalloproteinase(MMP)-2,apoptosis-related proteins Caspase-8,Caspase-3 and bcl-2 was detected by Western blotting.Results Transwell migration experiment results showed that the number of LNcap trans-membrane cells in ZD55-SATB1+DTX group was(28.20±3.19)cells after 24 h of intervention,which was significantly less than that in ZD55-SATB1 group[(39.60±4.98)cells](t=4.309,P<0.01),ZD55-EGFP group[(52.60±6.02)cells](t=8.001,P<0.01),DTX group[(65.50±5.12)cells](t=13.710,P<0.01)and PBS group[(106.60±5.18)cells](t=28.820,P<0.01).The Transwell invasion experiment results showed that the number of cells penetrating Matrigel gel into the lower compartment in ZD55-SATB1+DTX group[(23.60±3.58)cells]was significantly reduced as compared with the ZD55-SATB1 group[(32.40±5.08)cells](t=3.088,P<0.05),ZD55-EGFP group[(39.60±2.70)cells](t=7.610,P<0.01),DTX group[(44.60±4.62)cells](t=7.815,P<0.01)and PBS group[(98.20±4.32)cells](t=28.820,P<0.01).TUNEL results showed that apoptosis rate of each treatment group was higher than in PBS group[(5.92±0.90)%].The apoptosis rate in the combined group[(34.48±4.25)%]was significantly higher than in the ZD55-SATB1 group[(28.12±2.50)%](t=2.582,P<0.05),ZD55-EGFP group[(23.30±3.03)%](t=4.752,P<0.01),and DTX group[(22.35±2.44)%](t=4.956,P<0.01).Western blotting experiment results showed that in each treatment group,the expression of SATB1 protein was the lowest in ZD55-SATB1+DTX group,and the expression of E-cadherin protein was the highest when the protein expression of Vimentin and MMP-2 was the lowest.At the same time,the expression of Caspase-8 and Caspase-3 was the highest while bcl-2 was the lowest in the combined group and the difference was statistically significant when compared to single treatment group(P<0.05).The protein of E1A was normally expressed in the combined group,indicating that the combined use of DTX didn’t affect adenovirus replication and proliferation.Conclusion Oncolytic adenovirus ZD55-SATB1 combined with DTX can effectively inhibit the migration and invasion of LNcap cells and induce cell apoptosis in vitro,and the effect is better than ZD55-SATB1 or DTX alone.One of its mechanisms is to inhibit epithelial-mesenchymal transition(EMT)in tumor cells by up-regulation of E-cadherin,and down-regulation of Vimentin and MMP-2 when silencing SATB1 expression and inducing tumor apoptosis by up-regulating Caspase-8 and Caspase-3 and down-regulating bcl-2.