Selection of optimal antisense accessible sites of survivin and its application in treatment of gastric cancer.
OBJECTIVE
To select the optimal antisense accessible sites of survivin, a highly expressed gene in tumor tissues, in order to explore a novel approach to improve biological therapy of gastric cancer.


METHODS
The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcribed total survivin cRNA, then digested by RNase H. After primer extension and autoradiography, the antisense accessible sites (AAS) of survivin were selected. Then RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODNs) were synthesized and transferred into survivin highly- expressing gastric cancer cell line MKN-45. Survivin expression was detected by RT-PCR and Western Blotting. Cellular growth activities were assayed by tetrazolium bromide (MTT) colorimetry. Cellular ultrastructure was observed by electronic microscopy, while apoptosis was detected by annexin V-FITC and propidium iodide staining flow cytometry.


RESULTS
Thirteen AAS of survivin were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 207-226 bp, 187-206 bp, 126-145 bp and 44-63 bp of survivin cDNA respectively. When compared with non-tranfection controls, their corresponding AS-ODNs (AS-ODN(1), AS-ODN(2), AS-ODN(3) and AS-ODN(4)) could reduce Survivin mRNA levels in MKN-45 cells by 54.3+/-1.1% (t = 6.12, P<0.01), 86.1+/-1.0% (t = 5.27, P<0.01), 32.2+/-1.3% (t = 7.34, P<0.01) and 56.2+/-0.9% (t = 6.45, P<0.01) respectively, while survivin protein levels were decreased by 42.2+/-2.5% (t = 6.26, P<0.01), 75.4+/-3.1% (t = 7.11, P<0.01), 28.3+/-2.0% (t = 6.04, P<0.01) and 45.8+/-1.2% (t = 6.38, P<0.01) respectively. After transfection with 600 nmol/L AS-ODN1-AS-ODN(4) for 24 h, cell growth was inhibited by 28.12+/-1.54% (t = 7.62, P<0.01), 38.42+/-3.12% (t = 7.75, P<0.01), 21.46+/-2.63% (t = 5.94, P<0.01) and 32.12+/-1.77% (t = 6.17, P<0.01) respectively. Partial cancer cells presented the characteristic morphological changes of apoptosis, with apoptotic rates being 19.31+/-1.16% (t = 7.16, P<0.01), 29.24+/-1.94% (t = 8.15, P<0.01), 11.87+/-0.68% (t = 6.68, P<0.01) and 21.68+/-2.14% (t = 7.53, P<0.01) respectively.


CONCLUSIONS
The AAS of survivin could be effectively selected in vitro by random oligonucleotide library/RNase H cleavage method combined with computer software analysis, this has important reference values for further studying survivin-targeted therapy strategies for gastric cancer.
Tong
Qiang-Song
QS
Department of Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China. qs_tong@hotmail.com
Zheng
Li-Duan
LD
Chen
Fang-Min
FM
Zeng
Fu-Qing
FQ
Wang
Liang
L
Dong
Ji-Hua
JH
Lu
Gong-Cheng
GC
D017209:Apoptosis; D001483:Base Sequence; D002455:Cell Division; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics*; D016914:Ribonuclease H; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D000077022:Survivin
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D000077022:Survivin; D016914:Ribonuclease H