人端粒酶逆转录酶基因反义核酸结合位点的筛选

Screening of antisense accessible sites of human telomerase reverse transcriptase gene

目的筛选人端粒酶逆转录酶（hTERT）基因的反义核酸结合位点。方法合成20mer随机寡核苷酸文库，与体外转录的全长hTERTcRNA杂交，RNaseH切割后，经引物延伸、放射自显影，筛选出26个反义结合位点（AAS）。结合RNAstructure软件分析，选定具有显著茎环结构的7个位点作为最佳反义结合位点，合成其反义寡核苷酸（AS-ODN），转染前列腺癌细胞PC-3，运用噻唑蓝（MTT）比色及逆转录-聚合酶链反应（RT-PCR）分别对前列腺癌细胞生长抑制及hTERTmRNA表达情况进行检测。结果AS—ODN1—7转染前列腺癌细胞PC-3后，细胞生长受到明显抑制，其中AS-ODN3的抑制作用最明显（40．6±1．0）％，与各组抑制率差异有统计学意义（P〈0．05），hTERTmRNA表达水平亦受到明显抑制，亦以AS—ODN3的抑制作用最明显（35．8±1．2）％，与各组抑制率差异有统计学意义（P〈0．05）。结论筛选hTERT基因AAS并合成AS-ODN能有效封闭目的基因。

Objective To screen the antisense nucleic acid accessible sites of human telomerase reverse transcriptase （hTERT） gene. Methods The 20 met random oligonucleotide library was synthesized and then hybridized with in vitro transcripted total hTERT cRNA, then digested by RNase H. After primer extension and autoradiography, 25 antisense accessible sites （AAS） were selected. Seven AAS had obvious stem-loop structure after analyzed by the RNAstructure software. The seven AAS were considered as the best AAS and the complementary antisense oligonucleotide （AS-ODN） were synthesized and transferred into prostate cancer cell line PC-3. Tetrazolium bromide （MTF） colorimetry assay and reverse transcription polymerase chain reaction （RT-PCR） were used to detect the growth inhibition and expression level of hTERT mRNA in prostate cancer cells respectively. Results After AS-ODN1-7 was transferred into PC-3 cells, the AS-ODN3 could significantly inhibit the growth of PC-3 cells （40. 6 ± 1.0） % （P 〈0.05） , and suppress the hTERT mRNA expression （ 35.8 ± 1.2） % （ P 〈 0. 05 ） as compared with other groups. Conclusion To screen AAS of hTERT gene and synthesize there AS-ODAs can block the biological functions effectively.