个体性反义RNA对乳腺癌细胞突变p53基因表达的特异性封闭作用  

Targeted down-regulation of p53 gene expression by individual antisense RNA in vitro

作　　者：王亚红[1] 孙玉兰[1] 许少峰[1] 张媛媛[1] 张霖[1] 张彬[1] 冯玉梅[1] 牛瑞芳[1] 付丽[1] 

机构地区：[1]天津医科大学附属肿瘤医院乳腺病理研究室教育部乳腺癌防治重点实验室天津市肿瘤防治研究所乳腺病理研究室,300060

出　　处：《中华病理学杂志》2007年第8期544-549,共6页Chinese Journal of Pathology

基　　金：天津市科委基础研究重点基金(033801511)

摘　　要：目的研究针对 p53基因突变的个体性反义 RNA 对乳腺癌突变 p53基因的特异性封闭作用。方法通过免疫细胞化学 LSAB 法染色、聚合酶链反应-单链构象多态性分析及测序确定人乳腺癌细胞系 MDA-MB-231中 p53突变位点,构建针对该突变点的反义表达载体并制备其反义RNA。原位杂交技术检测反义 RNA 与细胞内突变 p53基因的特异性结合;在阳离子脂质体介导下将反义 RNA 转染细胞;以免疫细胞化学 LSAB 法染色及细胞生长抑制率观测反义 RNA 作用时效;Western blot 检测 p53蛋白的表达;四甲基偶氮唑盐法检测转染细胞的生长活性;流式细胞术检测转染细胞的细胞周期分布;原位缺口末端标记法检测转染细胞的凋亡情况。结果人乳腺癌细胞系MDA-MB-231的 p53第8外显子(exon8)有突变,据此位点构建反义表达载体[pGEM3zf(+/-)p53exon8]。原位杂交结果显示反义 RNA(ASp53exon8′RNA)在细胞质内有阳性杂交信号;于转染后48 h 反义 RNA 封闭效果最显著,突变 p53蛋白(mt-p53)的表达受抑制,细胞增殖能力下降,G_2/M 期阻滞。结论针对 p53基因突变位点制备的个体性反义 RNA 可特异性封闭乳腺癌细胞突变 p53基因的表达。Objective To investigate the effect of specific blockage of mutant p53 gene by individualized antisense RNA in vitro. Methods Mutation status of p53 in human breast cancer cell lines was determined by immunocytochemical staining, PCR-SSCP and sequencing. Single strand antisense transcription system targeting specific p53 mutation site （mt-p53） was constructed, and corresponding antisense RNA was prepared. The hybridization of antisense RNA with its corresponding mt-p53 gene was confirmed by in-situ hybridization. Human breast cancer ceils were transfected with antisense RNA by cationic liposome-mediated method. Time course of effects of antisense RNA was investigated by immunocytochemical staining and cell growth inhibiting assay. Expression of mt-p53 protein was examined by Western blot. Cell proliferation was evaluated by MTT assay and cell cycle distribution was determined by flow cytometry （FCM）. Apoptosis was determined by TUNEL assay. Results Mutation of p53 exon 8 was found in MDA-MB-231 cells and antisense transcription system （pGEM3zf（ ＋/- ）p53exon8）was then constructed successfully. In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. Fourth-eight hours after transfection, the antisense RNA （ASp53exon8RNA） had a significant retarding effect on p53 related proliferation inhibition, along with a decrease of p53 protein expression. Conclusions ASp53exon8＇RNA specifically blocks mt-p53 gene expression, resulting in an inhibition of MDA-MB-231 cell proliferation. Such an approach may be used as a therapeutic option against human malignancy.

关 键 词：乳腺肿瘤 基因 p53 RNA 反义 DNA突变分析 

分 类 号：R737.9[医药卫生—肿瘤]