[Construction of anti-sense cDNA library of human breast cancer cells during apoptosis induced by trichostatin A and preliminary screening of essential genes].
OBJECTIVE
To construct an anti-sense cDNA library of human breast cancer cells to screen essential genes with anti-tumor effects on apoptosis of human breast cancer cells induced by trichostatin A.


METHODS
Poly (A)(+)RNA was extracted from human breast cancer cells of the line MCF-7 treated by trichostatin A for 0, 12, 24, 36, 48, 60, or 72 h. cDNA were synthesized and inserted reversely into PCEP 4 vector to construct an anti-sense cDNA library. HeLa cells were transfected with the library DNA or blank PCEP 4 vector as control group. All the transfected cells were screened by 200 nmol/L trichostatin A and 200 microg/ml hygromycin B. Screening was stopped when the control cells died. Then the surviving cell clones were amplified and Hirt DNA was extracted. Several expressed sequence tags were thus obtained. The data were analyzed by bioinformatics and interested EST fragment was chosen for preliminary functional screening.


RESULTS
An anti-sense cDNA library was constructed containing 2 x 10(6) independent clones with an insert efficiency of more than 90%; DNA sequencing and bioinformatic analysis suggested that the No.27 survival clone was zinc transporter LIV1 showing a strong resistance against trichostatin A-induced apoptosis during functional screening.


CONCLUSIONS
An anti-sense cDNA library with high quantity and quality has been successfully constructed; LIV1 gene may be one of the essential genes with anti-tumor effects on apoptosis induced by trichostatin A.
Ma
Xiao-Li
XL
Cancer Biomedical Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Wang
Bei-Bei
BB
Wu
Peng
P
Lu
Yun-Ping
YP
Zhou
Jian-Feng
JF
Ma
Ding
D
D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D014162:Transfection
D018076:DNA, Complementary; D006877:Hydroxamic Acids; D011500:Protein Synthesis Inhibitors; C012589:trichostatin A