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一种特异性抗Ki-ras核酶在体外和细胞培养中的酶促作用及反义作用

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

作者信息

Giannini C D, Roth W K, Piiper A, Zeuzem S

机构信息

Medizinische Klinik II, Universitätsklinikum, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.

出版信息

Nucleic Acids Res. 1999 Jul 1;27(13):2737-44. doi: 10.1093/nar/27.13.2737.

DOI:10.1093/nar/27.13.2737
PMID:10373591
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC148483/
Abstract

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

摘要

由于其作用方式,核酶除了具有特定的切割活性外,还表现出反义效应。在本研究中,我们调查了锤头状核酶是否能够切割胰腺癌细胞系中突变的Ki-ras mRNA,以及反义效应是否有助于核酶的活性。设计了一种2'-O-烯丙基修饰的锤头状核酶,以特异性切割Ki-ras mRNA的突变形式(密码子12中的GUU基序)。通过RT-PCR监测Ki-ras RNA表达的活性,通过测定野生型与突变型Ki-ras mRNA的相对量、通过5-溴-2'-脱氧尿苷掺入监测细胞增殖以及通过在人胰腺腺癌细胞系CFPAC-1(该细胞系对Ki-ras突变杂合)的软琼脂中形成集落监测恶性程度。使用催化无活性的核酶作为对照,以区分反义活性和切割活性,并使用具有随机引导序列的核酶作为阴性对照。与催化无活性的核酶相比,具有催化活性的抗Ki-ras核酶在降低细胞Ki-ras mRNA水平、抑制细胞增殖和软琼脂中集落形成方面的效力至少高2倍。具有催化活性的抗Ki-ras核酶,但不是催化无活性或随机核酶,增加了CFPAC-1细胞中野生型与突变型Ki-ras mRNA的比例。总之,切割活性和反义效应都有助于具有催化活性的抗Ki-ras锤头状核酶的活性。特定的核酶可能有助于治疗在Ki-ras基因密码子12中含有致癌GTT突变的胰腺癌。

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