CAJ | 학술논문

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hTERT cRNA杂交,RNase H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hTERT mRNA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.
목적 사선인단립매역전록매(hTERT)기인적반의핵산결합위점.방법 합성20mer수궤과핵감산문고,여체외전록적전장hTERT cRNA잡교,RNase H절할후,경인물연신、방사자현영,사선출26개반의결합위점(AAS).결합RNAstructure연건분석,선정구유현저경배결구적7개위점작위최가반의결합위점,합성기반의과핵감산(AS-ODN),전염전렬선암세포PC-3,운용새서람(MTT)비색급역전록-취합매련반응(RT-PCR)분별대전렬선암세포생장억제급hTERT mRNA표체정황진행검측.결과 AS-ODN1-7전염전렬선암세포PC-3후,세포생장수도명현억제,기중AS-ODN3적억제작용최명현(40.6±1.0)％,여각조억제솔차이유통계학의의(P＜0.05),hTERTmRNA표체수평역수도명현억제,역이AS-ODN3적억제작용최명현(35.8±1.2)％,여각조억제솔차이유통계학의의(P＜0.05).결론 사선hTERT기인AAS병합성AS-ODN능유효봉폐목적기인.
Objective To screen the antisense nucleic acid accessible sites of human telomerase reverse transcriptase (hTERT) gene.Methods The 20 mer 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,26 antisease 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 (MTT) 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.