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首页> 外文期刊>World Journal of Gastroenterology >Selection of optimal antisense accessible sites of survivin and its application in treatment of gastric cancer.
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Selection of optimal antisense accessible sites of survivin and its application in treatment of gastric cancer.

机译：survivin最佳反义可及位点的选择及其在胃癌治疗中的应用。

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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.
机译：目的：选择在肿瘤组织中高表达的基因survivin的最佳反义可及位点，以探索改善胃癌生物治疗的新方法。方法：合成20聚体随机寡核苷酸文库，与体外转录的总生存素cRNA杂交，然后用RNase H酶切。引物延伸和放射自显影后，选择生存素的反义可及位点（AAS）。然后使用RNADraw软件分析和选择具有明显茎环结构的AAS，据此合成互补的反义寡核苷酸（AS-ODNs），并将其转移到高表达survivin的胃癌细胞系MKN-45中。通过RT-PCR和蛋白质印迹检测存活蛋白的表达。细胞生长活性通过溴化四唑（MTT）比色法测定。电镜观察细胞超微结构，膜联蛋白V-FITC和碘化丙啶染色流式细胞仪检测细胞凋亡。结果：体外选择了13种存活蛋白。选择了四个具有茎环结构的AAS，分别位于survivin cDNA的207-226 bp，187-206 bp，126-145 bp和44-63 bp。与非转染对照相比，它们对应的AS-ODN（AS-ODN（1），AS-ODN（2），AS-ODN（3）和AS-ODN（4））可以降低MKN-中Survivin mRNA的水平45个细胞，分别为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）和56.2 +/- 0.9％（t = 6.45，P <0.01），而survivin蛋白水平分别降低了42.2 +/- 2.5％（t = 6.26，P <0.01），75.4 +/- 3.1％（t =分别为7.11，P <0.01），28.3 +/- 2.0％（t = 6.04，P <0.01）和45.8 +/- 1.2％（t = 6.38，P <0.01）。用600 nmol / L AS-ODN1-AS-ODN（4）转染24小时后，细胞生长被抑制28.12 +/- 1.54％（t = 7.62，P <0.01），38.42 +/- 3.12％（t分别为7.75，P <0.01），21.46 +/- 2.63％（t = 5.94，P <0.01）和32.12 +/- 1.77％（t = 6.17，P <0.01）。部分癌细胞呈现凋亡的形态学特征变化，凋亡率分别为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）和21.68 +/- 2.14％（t = 7.53，P <0.01）。结论：随机寡核苷酸文库/ RNase H裂解方法结合计算机软件分析可以有效地选择survivin的AAS，为进一步研究以survivin为靶标的胃癌治疗策略提供了重要的参考价值。

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