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.coli TOP10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24 h比48 h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.