Aim: 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.
Conclusion: 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.