pSilencer/AMACR干扰载体的构建及转染人前列腺癌PC-3细胞  

Construction of AMACR RNAi vectors and transfection into human PC-3 cell line

作　　者：袁建林[1] 王立国[1] 于磊[1] 张更[1] 武国军[1] 

机构地区：[1]第四军医大学西京医院泌尿外科,西安710032

出　　处：《山西医科大学学报》2008年第12期1071-1074,共4页Journal of Shanxi Medical University

基　　金：陕西省自然科学基金国际合作基金资助项目(2005KW-15)

摘　　要：目的通过RNAi封闭前列腺癌PC-3细胞系α-甲酰辅酶A消旋酶(AMACR)的表达。方法根据AMACR基因序列设计并合成两对siRNA,插入pSilencer 4.1-CMV neo载体。用脂质体将pSilencer/AMACR和对照空载体pSilencer转染前列腺癌PC-3细胞,通过RT-PCR和Western blot检测转染细胞的AMACR表达情况。结果成功构建了AMACR RNAi真核表达载体,利用脂质体转染PC-3细胞后,RT-PCR和Western Blot鉴定结果显示:AMACR的表达水平显著降低。结论AMACR RNAi真核表达载体在mRNA和蛋白水平阻断了AMACR的表达。Objective To down-regulate the expression of α-methylacyl-CoA-racemase（AMACR）in PC-3 cell line by RNA interference（RNAi）technology. Methods Vector capable of producing hairpin siRNA molecule for AMACR was constructed using the mammalian expression plasmid vector pSilencer 4.1-CMV neo. Using LipofectAmineTM 2000 reagent, the human prostate cancer cell line PC-3 was transfected with RNAi eukaryotic expression vectors pSileneer/AMACR and control plasmid pSilencer. RT-PCR and Western blot were used to testify the mRNA and protein level in the transfected and parental PC-3 cells. Results The vectors were successfully constructed and confirmed by DNA sequencing. PSilencer/AMACR or pSilencer was stably transfected into prostate cancer PC-3 cells using LipofectAmine^TM 2000 reagent. RT-PCR and Western blot showed that expression of AMACR mRNA and protein significantly decreased in the pSilencer/AMACR1 and pSilencer/AMACR2 transfected PC-3 cells. While in control plasmid pSilencer transfected PC-3 cells,AMACR expression remained the same level as the parental cells. Conclusion AMACR expression could be inhibited by siRNA transfectants in PC-3 cells at mRNA and protein levels.

关 键 词：前列腺肿瘤 AMACR RNA干扰 

分 类 号：R392.11[医药卫生—免疫学]