沉默乙酰肝素酶基因对人卵巢癌SKOV3细胞增殖、侵袭能力的影响  

Silencing heparanase suppresses proliferation and invasion in ovarian cancer SKOV3 cells

作　　者：赵玲[1] 杨鹰[1] 

机构地区：[1]第三军医大学新桥医院妇产科,重庆400037

出　　处：《第三军医大学学报》2011年第22期2361-2365,共5页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC2008BB122)~~

摘　　要：目的观察沉默乙酰肝素酶(heparanase,HPA)基因对人卵巢癌SKOV3细胞的增殖及侵袭能力的影响。方法构建针对HPA基因的shRNA慢病毒载体,感染人卵巢癌SKOV3细胞。实验设计分为未转染组、实验转染组(选择3个靶点设计HPA-shRNA-1、HPA-shRNA-2、HPA-shRNA-3序列)、空白转染组;转染后,采用荧光定量PCR、Western blot方法检测HPA在基因水平和转录后蛋白水平的沉默效率;使用流式细胞仪检测细胞周期变化,CCK-8法检测细胞增殖情况,基质凝胶侵袭实验检测细胞侵袭能力变化。结果①构建的shRNA慢病毒载体感染卵巢癌SKOV3细胞后,见HPA-shR-NA-1和HPA-shRNA-2序列的HPA基因和蛋白水平均显著下降(P<0.05),而HPA-shRNA-3序列在其表达上无降低,为无效转染序列;②实验转染组中有效转染序列HPA-shRNA-1、HPA-shRNA-2组中均见细胞G1期比例增加[分别为(69.69±3.87)%、(66.29±4.06)%],细胞增殖受到抑制[分别为(1.77±0.13)、(1.74±0.27)],穿膜细胞数显著减少[分别为(22.40±5.02)、(23.42±5.72)],与未转染组及空白转染组之间相比有明显差异(P<0.05)。结论干扰HPA基因后,对卵巢癌细胞的增殖及侵袭能力有明显的抑制作用,其作用机制与HPA的基因和蛋白表达下调有关。Objective To explore the effect of silencing heparanase gene by RNA short hairpin on the proliferation and invasion in human ovarian cancer cell line SKOV3.Methods After shRNA lentiviral vectors targeting heparanase gene was constructed,they were transfected into SKOV3 cells.Experimental transfection groups included the interference sequences HPA shRNA-1 group,HPA shRNA-2,and HPA shRNA-3 group,and the untransfected group amd blank transfection group served as control.After transfection,the interference efficiency was observed though fluorescence quantitative PCR and Western blotting to detect the expression of heparanase at mRNA and protein levels.Flow cytometry was employed to test cell cycle changes,cell counting kit-8（CCK-8） for the detection of cell proliferation,and matrix gel invasion assays for the detection of cell invasion ability.Results After shRNA lentiviral vectors were transfected into SKOV3 cells,HPA expression was significantly decreased at mRNA and protein levels in the HPA-shRNA-1 sequence and the HPA-shRNA-2 sequence groups（P0.05）,whereas the expression levels of HPA-shRNA-3 sequence group was not decreased,indicating it was an ineffective sequence.In the SKOV3 cells transfected with effective sequences HPA-shRNA-1 and HPA-shRNA-2,the cell percentage of G1 phase was decreased,the proliferation（OD index） was significantly decreased and transmembrane cell number was increasingly decreased.There was a significantly difference between the experimental groups and untransfected and blank transfection groups respectively（P0.05）.Conclusion Silencing HPA by RNA interference inhibits heparanase expression,and suppresses cell proli-feration and invasion efficiently,which may be correlated with the downregulation of HPA gene and protein.

关 键 词：沉默基因 乙酰肝素酶 卵巢癌 肿瘤浸润 

分 类 号：R394.3[医药卫生—医学遗传学] R73-36[医药卫生—基础医学]