Construction of the antisense eukaryotic vector for proliferating cell nuclear antigen gene and its expression in bladder cancer EJ cell line.
To explore a novel strategy for antisense gene therapy of cancer, the coding sequence of human proliferating cell nuclear antigen (PCNA) cDNA was reversely inserted into the eukaryotic vector pLXSN by molecular cloning techniques and transferred into bladder cancer EJ cells with liposome. The PCNA expression in transferred cells was dynamically detected by immunofluorescence and RT-PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorimetric and cloning formation methods. In the experiment, the antisense eukaryotic vector was successfully constructed and named as pLAPSN. After transfection with it for 1-7 days, PCNA protein and mRNA levels in cancer cells were blocked by 16.74%-84.21% (P < 0.05) and 23.27%-86.15% (P < 0.05) respectively. The proliferation activities of transferred cells were inhibited by 27.91%-62.07% (P < 0.01), with cloning formation abilities being decreased by 50.81% (P < 0.01). It was concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique, which could serve as an ideal strategy for gene therapy of bladder cancer.
Tong
Qiangsong
Q
Department of Urology, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022.
Zeng
Fuqing
F
Qi
Yipeng
Y
Zhu
Zhaohui
Z
Lu
Gongcheng
G
D002455:Cell Division; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004273:DNA, Neoplasm / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000235:genetics*; D016372:RNA, Antisense / Q000235:genetics*; D014162:Transfection; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D018809:Proliferating Cell Nuclear Antigen; D016372:RNA, Antisense