中文摘要：

目的 构建STK15基因特异的RNA干扰（RNAi）真核表达质粒,将其转染人结肠癌细胞SW480后,观察对STK15基因表达的抑制作用及对肿瘤细胞侵袭能力的影响.方法 设计并合成能表达小发夹结构RNA （shRNA）的DNA序列,退火后与载体pGPU6/GFP/Neo连接,构建重组表达载体pGPU6/GFP/NeoSTK15 shRNA,转化DH5α菌株后挑选阳性克隆,提取质粒进行序列测定.脂质体介导重组质粒转染人结肠癌SW480细胞,于转染后24h和48 h通过反转录-聚合酶链反应（RT-PCR）和蛋白印迹法分别在mRNA和蛋白水平检测STK15基因的表达,并通过细胞体外侵袭实验检测肿瘤细胞的侵袭能力.结果 测序证实插入pGPU6/GFP/Neo载体的DNA序列、方向和位点均正确.与阴性质粒转染组比较,pGPU6/GFP/Neo-STK15 shRNA质粒转染细胞24h和48 h后,STK15基因的mRNA和蛋白表达水平均明显降低,且呈现时间依赖性,STK15 mRNA水平分别下调80％和98％,蛋白水平分别下调40％和80％.与阴性质粒转染组比较,pGPU6/GFP/Neo-STK15shRNA质粒转染细胞的侵袭能力分别下降约67％和89％.结论 成功构建了针对STK15基因的特异性shRNA真核表达载体,转染细胞后可抑制STK15基因的表达和细胞的侵袭能力,为进一步研究STK15基因功能以及探讨结肠癌治疗的新途径奠定了基础.

英文摘要：

Objective To construct the STK15 eukaryotic expression vector for RNA interference and investigate its inhibitory effect on STK15 gene expression and invasion of human colon cancer SW480 cells following transfection.Methods The oligonucleotide sequences encoding short hairpin RNA （shRNA） were designed and synthesized,which were annealed and cloned into vector pGPU6/GFP/Neo for construction of pGPU6/GFP/Neo-STK15 shRNA,the recombinant plasmid,which was subsequently transformed into the cell strain DHSα for selection of the positive clones.This entailed extraction and sequencing of the recombinant plasmids.The recombinant plasmid was then transfected into human colon cancer SW480 cells via liposomes.This was followed by assay of the STK15 gene expression products at mRNA and protein levels by using reverse transcriptase-polymerase chain reaction （RT-PCR）and Western blotting at 24 and 48 hours after transfection.And the invasive properties were examined by in vitro invasion assay.Results Gene sequencing warranted the sequence,location and direction of transfection with pGPU6/GFP/Neo vector.Compared with negatively transfected plasmids,the pGPU6/GFP/Neo-STK15 shRNA yielded markedly attenuated levels of STK15 mRNA transcription and protein in SW480 cells,which appeared in a time-dependent manner,at 24 and 48 hours after transfection.Compared with control group,the STK15 mRNA expression was downregulated by 80％ and 98％ and protein expression by 40％ and 80％ at 24 and 48 hours following transfection,respectively.The invasion capacity of tumor cells was reduced by 67％ and 89％ at 24 and 48 hours after transfection,respectively.Conclusion The successfully constructed eukaryotic expression vector specifically for RNA interference STK15 gene inhibits STK15 expression and invasion of SW480 cells,thus providing a novel approach for further studying the function of STK15 and therapeutic direction of colon cancer.