构建survivin shRNA重组载体及靶向下调PC3细胞survivin mRNA的表达

背景：survivin基因特异性表达于肿瘤和胚胎组织,与肿瘤细胞的分化增殖、浸润转移以及多药耐药密切相关。目的：构建靶向survivin基因的shRNA重组质粒表达载体,转染前列腺癌细胞PC3,验证该载体能否下调细胞survivin基因mRNA水平。方法：以survivin基因为靶点设计具有短发夹结构的shRNA序列,经退火成互补双链后克隆入pENTR/U6建立重组表达载体pENTR/U6-SUR;转化E.coliTOP10菌株,挑取阳性菌落进行菌落PCR和测序鉴定;将重组质粒转染前列腺癌细胞株PC3细胞,RT-PCR检测重组质粒对细胞survivin基因mRNA水平的抑制效果。结果与结论：将设计合成的shRNA序列经退火后克隆至pENTR/U6载体中,菌落PCR可扩增出目的条带,测序结果证实插入片段为所需序列;pENTR/U6-SUR重组质粒转染后PC3细胞survivin基因mRNA表达水平显著下降,且24h比48h作用更明显。成功构建了靶向survivin基因的shRNA质粒表达载体,并证实该载体显著下调了PC3细胞中survivin基因mRNA水平。

Construction of survivin shRNA recombinant expression vector and downregulation of survivin mRNA expression in PC3 cells

BACKGROUND：survivin mRNA is specifically expressed in tumor and embryonic tissue and is closely related to differentiation,proliferation,infiltration and metastasis of tumor cells as well as multidrug resistance.OBJECTIVE：To construct the expression vector of small hairpin RNA（shRNA） targeting human survivin gene and detect the effectiveness of gene silencing in PC3 cells（human prostate cancer cell line）.METHODS：Two single-stranded DNA oligonucleotides for shRNA expression targeted survivin gene were chemically synthesized.The top and bottom strand oligos were annealed to generate a double-stranded oligonucleotide（ds oligo）.The the oligo was cloned into pENTR/U6 expression vector（pENTR/U6-SUR）,and then PCR and sequencing analyses were conducted to verify the constructs.After transfecting the verified plasmids into PC3 cells,RT-PCR was performed to determine the mRNA level of survivin gene.RESULTS AND CONCLUSION：PCR and sequencing analyses demonstrated that shRNA template targeting survivin gene had been inserted at the expected site and the insertion sequence was perfectly corrected.The RT-PCR results showed that survivin expression in PC3 was downregulated at mRNA level.The recombinant plasmid had a better affect at 24 hours than 48 hours.The shRNA expression vector targeting survivin gene has been constructed successfully,and it would be a useful method to develop specific survivin-silencing therapeutics in further gene therapy study.