Potent subunit-specific effects on cell growth and drug sensitivity from optimised siRNA-mediated silencing of ribonucleotide reductase.

Glen Reid, Natacha Coppieters 't Wallant, Rachna Patel, Ana Antonic, Faamatala Saxon-Aliifaalogo, Helen Cao, Gill Webster, James D Watson
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Abstract

Ribonucleotide reductase (RR) has an essential role in DNA synthesis and repair and is a therapeutic target in a number of different cancers. Previous studies have shown that RNAi-mediated knockdown of either the RRM1 or RRM2 subunit sensitizes cells to the cytotoxic effects of the nucleoside analogs and more recently it has been shown that RRM2 knockdown itself has a growth inhibitory effect. Here we compare the effects of siRNA-mediated knockdown of both RRM1 and RRM2 subunits of RR in A549 and HCT-116 cells using an optimized transfection protocol. Growth of A549 cells was strongly inhibited by efficient siRNA-mediated silencing of either RRM1 or RRM2, and knockdown of each subunit led to long-term growth inhibition and cell-cycle arrest. Knockdown with sub growth inhibitory siRNA concentrations sensitized A549 and HCT-116 cells to gemcitabine when RRM1 was targeted, whereas RRM2 knockdown led to hydroxyurea sensitization. These results suggest that the inhibition of cell growth, rather than drug sensitization, is the major effect of RRM1 and RRM2 knockdown. In an A549 xenograft model, cells transfected with RRM1-specific siRNA failed to form tumors in 6 out of 8 CD1 nude mice, whereas those transfected with RRM2-specific siRNA grew but at a reduced rate. Taken together, these data demonstrate that siRNA-mediated knockdown of the RRM1 subunit is more effective than knockdown of RRM2 in inhibiting the growth of cancer cell lines and suggest that RRM1 is a potential target for nucleic acid-based cancer therapies, either alone or in combination with gemcitabine.

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优化sirna介导的核糖核苷酸还原酶沉默对细胞生长和药物敏感性的有效亚单位特异性影响。
核糖核苷酸还原酶(RR)在DNA合成和修复中起着重要作用,是许多不同癌症的治疗靶点。先前的研究表明,rnai介导的RRM1或RRM2亚基的敲低使细胞对核苷类似物的细胞毒性作用敏感,最近的研究表明,RRM2敲低本身具有生长抑制作用。在这里,我们使用优化的转染方案比较了sirna介导的RRM1和RRM2亚基在A549和HCT-116细胞中的作用。通过sirna介导的RRM1或RRM2的有效沉默,A549细胞的生长受到强烈抑制,并且每个亚基的敲低导致长期生长抑制和细胞周期停滞。当RRM1为靶点时,用亚生长抑制siRNA浓度敲低A549和HCT-116细胞对吉西他滨增敏,而RRM2敲低导致羟基脲增敏。这些结果表明,RRM1和RRM2敲低的主要作用是抑制细胞生长,而不是药物致敏。在A549异种移植模型中,转染rrm1特异性siRNA的细胞在8只CD1裸鼠中有6只不能形成肿瘤,而转染rrm2特异性siRNA的细胞生长但速度减慢。综上所述,这些数据表明,sirna介导的RRM1亚基敲低比敲低RRM2在抑制癌细胞系生长方面更有效,并表明RRM1是基于核酸的癌症治疗的潜在靶点,无论是单独使用还是与吉西他滨联合使用。
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