Mao Li-Jun, Zheng Jun-Nian, Li Wang, Wang Jun-Qi, Chen Jia-Cun, Sun Xiao-Qing
Department of Urology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, China.
Zhonghua Nan Ke Xue. 2010 Aug;16(8):679-83.
To construct an oncolytic adenovirus with the DD3 promoter regulation, expressing small hairpin RNA and targeting the SATB1 gene (SATBI-shRNA), and to evaluate its potential for inhibiting the growth of human prostatic carcinoma cells (LNCaP) in vitro.
SATB1-shRNA expression cassettes containing the H1 promoter were produced by PCR from pSilencer3. 1-SATB1 and inserted into the pZD55 vector, and the recombinant plasmid pZD55-SATB1-shRNA was constructed, pZD55SATB1-shRNA and pZXC2-DD3-E1A were digested with EcoRV and Xba I , and the obtained expression cassettes linked each other to construct the recombinant plasmid pDD3-ZD55-SATB1, which was cotransfected with the pBHGE3 packaging plasmids mixture into 293 cells by Effectence. The recombined adenoviruses DD3-ZD55-SATB1 were identified by PCR. Viruses were propagated on HEK293 cells and purified by standard techniques, and the functional PFU titers determined by plaque assay on 293 cells. The antitumor potential of DD3-ZD55-SATB1 to LNCaP was evaluated by the crystal violet dye method. The expression level of the E1A gene was detected by Western blot, and that of the SATB1 gene in the adenovirus-infected LNCaP cells by both Western blot and RT-PCR.
An oncolytic adenovirus expressing SATB1-shRNA with the DD3 promoter regulation, DD3-ZD55-SATB1, was constructed successfully, whose functional PFU titer was 1.2 x 10(10) PFU/ml. DD3-ZD55-SATB1 showed an obvious cytopathic effect and a selective expression of E1A in the adenovirus-infected LNCaP cells; it exhibited a high LNCaP-targetability and specific SATB1-silencing effect.
The successful construction of the oncolytic adenovirus DD3-ZD55-SATB1 offers a new tool for researches on the gene therapy for human prostate cancer.
构建一种由DD3启动子调控、表达小发夹RNA并靶向SATB1基因的溶瘤腺病毒（SATBI-shRNA），并评估其体外抑制人前列腺癌细胞（LNCaP）生长的潜力。
从pSilencer3.1-SATB1通过PCR制备含H1启动子的SATB1-shRNA表达盒，并插入pZD55载体，构建重组质粒pZD55-SATB1-shRNA。用EcoRV和Xba I消化pZD55SATB1-shRNA和pZXC2-DD3-E1A，将获得的表达盒相互连接构建重组质粒pDD3-ZD55-SATB1，通过Effectence将其与pBHGE3包装质粒混合物共转染至293细胞。通过PCR鉴定重组腺病毒DD3-ZD55-SATB1。病毒在HEK293细胞上扩增并通过标准技术纯化，通过在293细胞上的噬斑测定确定功能性PFU滴度。用结晶紫染色法评估DD3-ZD55-SATB1对LNCaP的抗肿瘤潜力。通过蛋白质免疫印迹法检测E1A基因的表达水平，通过蛋白质免疫印迹法和逆转录-聚合酶链反应检测腺病毒感染的LNCaP细胞中SATB1基因的表达水平。
成功构建了由DD3启动子调控、表达SATB1-shRNA的溶瘤腺病毒DD3-ZD55-SATB1，其功能性PFU滴度为1.2×10(10) PFU/ml。DD3-ZD55-SATB1在腺病毒感染的LNCaP细胞中表现出明显的细胞病变效应和E1A的选择性表达；它表现出高LNCaP靶向性和特异性SATB1沉默效应。
溶瘤腺病毒DD3-ZD55-SATB1的成功构建为人类前列腺癌基因治疗研究提供了一种新工具。
