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[通过特殊小干扰RNA敲低PLK1信使核糖核酸]

[Knockdown of PLK1 mRNA by special siRNA].

作者信息

Chen Gong-Xing, Dong Qing-Hua, Zhang Jia-Wei, Fu Fang-Fang, Xu Ze-Feng, Liang Qiao-Yi, Zheng Shu, Ding Jia-Yi

机构信息

Cancer Institute, The 2nd Affiliated Hospital, Medical college, Zhejiang University, Hangzhou 310009, China.

出版信息

Yi Chuan. 2006 Jan;28(1):21-5.

Abstract

To study knockdown effect of small interfering RNA (siRNA) to PLK1 (Polo-like kinase 1) mRNA in colorectal cancer cell line SW480 and its mitosis and growth was changed. Ten special siRNA molecules were designed targeting different sites of PLK1 mRNA sequence and chemically synthesized. The siRNA molecules were transfected into SW480 by Oligofectamine. The gene mRNA level was assayed by Real-Time PCR. The changes of PLK1 protein, SW480 cell cycle and survival percentage was checked by Western-blot, Flow cytometry and Cell counter assays respectively. All 10 siRNA molecules knocked PLK1 mRNA down in different level. Of them P1, P4 and P9 showed over 80% knockdown efficiency and the others had more than 20% knockdown efficiency to PLK1 mRNA. The best knockdown effect over 95% of all groups was at 25 nmol/L of a mixture with P1, P4 and P9 siRNA equally. In this situation the protein was very less and the cells were blocked at G2 phase of cell cycle. After 72 h cell survival percentages were consistent with PKL1 mRNA level change by siRNA gradient concentration. The results showed that siRNA targeting PLK1 mRNA had effectively knocked PLK1 mRNA down in SW480 cell line. And a blended siRNAs held the best knockdown effect. The cell was blocked on the mitosis and growth.

摘要

研究小干扰RNA(siRNA)对结直肠癌细胞系SW480中PLK1(Polo样激酶1)mRNA的敲低作用及其对细胞有丝分裂和生长的影响。针对PLK1 mRNA序列的不同位点设计并化学合成了10种特异性siRNA分子。通过脂质体转染试剂将siRNA分子转染至SW480细胞中。采用实时荧光定量PCR检测基因mRNA水平。分别通过蛋白质免疫印迹法、流式细胞术和细胞计数法检测PLK1蛋白、SW480细胞周期及细胞存活率的变化。所有10种siRNA分子均能不同程度地敲低PLK1 mRNA。其中,P1、P4和P9的敲低效率超过80%,其他分子对PLK1 mRNA的敲低效率超过20%。所有组中敲低效果最佳(超过95%)的是25 nmol/L由P1、P4和P9 siRNA等比例混合而成的混合物。在此条件下,蛋白表达量极低,细胞被阻滞在细胞周期的G2期。72小时后,细胞存活率与siRNA梯度浓度引起的PKL1 mRNA水平变化一致。结果表明,靶向PLK1 mRNA的siRNA能有效敲低SW480细胞系中的PLK1 mRNA。混合siRNA具有最佳敲低效果,细胞的有丝分裂和生长受到阻滞。

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