摘要
目的研究荷载精子相关抗原9（sperm associated antigen 9，SPAG9）基因shRNA的溶瘤腺病毒对人前列腺癌细胞株PC-3和DU145增殖及侵袭作用的影响，并初步探讨其作用机制。方法采用同源重组法构建溶瘤腺病毒ZD55-SPAG9，采用PCR法进行鉴定。荧光显微镜观察病毒转染情况。CCK-8法检测溶瘤腺病毒对细胞增殖的作用。结晶紫法观察溶瘤腺病毒的细胞毒性。Transwell检测溶瘤腺病毒对细胞侵袭作用的影响。Western blot检测溶瘤腺病毒处理后PC-3和DU145细胞中SPAG9、E-cadherin、Vimentin和MMP-2蛋白的表达。结果溶瘤腺病毒ZD55-SPAG9包装成功，在PC-3和DU145细胞转染情况良好。ZD55-SPAG9对前列腺癌细胞增殖的抑制作用呈剂量和时间依赖性。随着病毒滴度的增加，ZD55-SPAG9对PC-3和DU145细胞的毒性逐渐增加。与PBS组和ZD55-EGFP组比较，ZD55-SPAG9组侵袭的PC-3和DU145细胞数目明显减少，差异有统计学意义（P〈0．01）。与PBS组和ZD55-EGFP组比较，ZD55-SPAG9组PC-3和DUl45细胞中SPAG9、MMP-2、Vimentin蛋白表达量显著降低，E-cadherin蛋白表达升高，差异有统计学意义（P〈0．05）。结论ZD55-SPAG9能够显著抑制人前列腺癌PC-3和DU145细胞的增殖和侵袭能力，其机制可能与上皮细胞间质化有关。
Objective To observe the effects of oncolytic adenovirus carrying SPAG9 gene on the proliferation and invasion of prostate cancer PC - 3 and DU145 cells. Methods Oncolytic adenovirus carrying SPAG9 gene （ ZD55 - SPAG9） was constructed and identified by PCR. The transfection efficiency of adenovirus was detected with fluorescence microscopy. The proliferation of PC - 3 and DU145 cells after exposed to ZD55 - SPAG9 was assessed by CCK - 8 assay. The cytotoxic effects of adenoviruses ZD55 - SPAG9 to PC - 3 and DU145 cells were detected by crystal violet staining. The invasion capacities of PC - 3 and DU145 cells were detected by Transwell assay. The expression of SPAG9 and invasion associated protein MMP -2, Vimentin and E -cadherin in PC -3 and DU145 cells were detected by Western blot. Results Adenovirus ZD55 - SPAG9 was successfully constructed. ZD55 - SPAG9 could inhibit the proliferation of PC - 3 and DU145 cells in a dose and time dependent manner. Also, ZD55 -SPAG9 had the strongest cytotoxicity in PC -3 and DU145 cells compared with ZD55 -EGFP. Compared with the ZD55 -EGFP and PBS groups, the ZD55 -SPAG9 group showed a remarkably declined number of invaded PC - 3 and DU145 （P 〈 0.01 ）. Furthermore, ZD55 - SPAG9 treatment resulted in remarkably increased amounts of SPAG9, MMP - 2 and Vimentin and a decreased amount of E - cadherin, compared with exposure to BS and ZD55 - EGFP （P 〈 0.05）. Conclusions ZD55 - SPAG9 can inhibit the proliferation and invasion of PC - 3 and DU145 cells, which may be associated with epithelial - mesenchymal transition.
作者
丁猛
潘俊
于海元
毛立军
DING Meng;PAN Jun;YU Haiyuan;MAO Lijun(Graduate School,Xuzhou Medical University,Xuzhou,Jiangsu 221004,China;Deparment of Urinary Surgery,the Affiliated Hospital of Xuzhou Medical University,Xuzhou,Jiangsu 221002)
出处
《徐州医科大学学报》
CAS
2018年第6期357-362,共6页
Journal of Xuzhou Medical University
基金
江苏省自然科学基金（BK20151166）
江苏省普通高校研究生科研创新计划项目（KYZZ16-0403）
关键词
精子相关抗原9
溶瘤腺病毒
前列腺癌
侵袭作用
sperm associated antigen 9
oncolytic adenovirus
prostate cancer
cell invasion
