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合成 2 0mer随机寡核苷酸文库 ,与体外转录出的全长survivincRNA杂交 ,RNaseH酶切割后 ,经引物延伸、放射自显影 ,共筛选出 13个针对survivin基因的反义结合位点 (antisenseaccessiblesites ,AAS) .运用RNADraw软件分析、选定具有显著茎环结构的 4个位点 ,合成互补性反义寡核苷酸AS ODN1、AS ODN2 、AS ODN3 、AS ODN4并转染高表达survivin基因的胃癌细胞株MKN 4 5 .逆转录聚合酶链反应和Western印迹检测发现MKN 4 5细胞的survivinmRNA和蛋白水平均有显著的下降 ;MTT比色法证实 6 0 0nmol LAS ODN1～AS ODN4转染 2 4h后细胞生长受到明显抑制 ,透射电镜、annexinⅤ FITC和PI双染色流式细胞术均检测到细胞凋亡 .说明运用随机寡核苷酸文库 RNaseH酶切割与计算机分析相结合的方法 ,在体外有效筛选出survivin的反义核酸结合位点 ,其相应的反义寡核苷酸能阻断survivin基因的生物学功能 .
A total of 20 random oligonucleotide libraries were synthesized and hybridized with the full-length survivincRNA transcribed in vitro. After cleavage by RNaseH, 13 antisense accessible sites were screened for the survivin gene by primer extension and autoradiography. AAS) .According to RNADraw software, 4 sites with significant stem-loop structure were selected to synthesize complementary antisense oligodeoxynucleotides AS ODN1, AS ODN2, AS ODN3 and AS ODN4 and transfected gastric cancer with high expression of survivin gene Cell line MKN 4 5. The mRNA and protein levels of survivin in MKN 4 5 cells were significantly decreased by reverse transcription polymerase chain reaction and Western blotting. MTT assay confirmed that the transfection of 600 nm LAS ODN1 ~ AS ODN4 for 24 h Cell growth was significantly inhibited, and apoptosis was detected by transmission electron microscopy, annexin Ⅴ FITC and PI double staining flow cytometry, indicating that the combination of random oligonucleotide library RNaseH cleavage and computer analysis could effectively screen out The antisense nucleic acid binding site of survivin, its corresponding antisense oligonucleotide can block the biological function of survivin gene.