靶向Pin1基因的shRNA干扰质粒的构建及其对大肠癌SW620细胞Pin1基因的抑制作用

Construction of shRNA Interfering Plasmid Targeting Pin1 Gene and Its Inhibitory Effect on Pin1 Gene of Colon Cancer SW620 Cells

目的构建靶向Pin1基因的短发夹RNA(shRNA)干扰真核表达质粒,并检测其对大肠癌SW620细胞Pin1基因的抑制作用。方法设计合成特异性针对人Pin1基因的寡核苷酸序列,退火后与pGenesil-1载体连接,构建靶向Pin1基因的shRNA真核表达质粒pGenesil-1-Pin1(+),利用脂质体转染大肠癌SW620细胞,以质粒pGenesil-1-Pin(-)转染细胞和未转染细胞为对照。利用荧光显微镜观察转染后细胞表达的绿色荧光蛋白,估算转染效率;应用实时荧光定量PCR和Western blot检测其对SW620细胞Pin1基因mRNA转录及蛋白表达的影响。结果所构建的特异Pin1shRNA真核表达质粒经酶切及测序证明构建正确,转染SW620细胞的转染效率为64%。shRNA对SW620细胞Pin1基因mRNA的抑制率在转染后72h最高,为70.2%,蛋白抑制率为60%。结论已成功构建了靶向Pin1基因的shRNA干扰真核表达质粒,可抑制SW620细胞Pin1基因mRNA的转录及蛋白的表达。

Objective To construct the short hairpin RNA（shRNA） interfering plasmid targeting Pin1 gene and determine its inhibitory effect on the Pinl gene of colon cancer SW620 cells. Methods The oligonueleotide sequence specific to human Pinl gene was designed and synthesized then, after annealing, inserted into vector pGenesil-1. The constructed shRNA eukaryotic expression vector pGenesil-1-Pinl （＋） was transfeeted to SW620 cells in mediation of liposome, using the SW620 cells transfected with plasmid pGenesil-l-Pinl（-） and the untransfected SW620 cells as controls. The green fluorescent protein （GFP） expressed in transfeeted cells were observed by fluorescent microscopy, based on which the transfection efficacy was calculated. The effects of plasmid pGene- sil-l-Pin 1 （＋） on the transcription of Pin1 mRNA and expression of Pin l protein in SW620 cells were determined by real-time fluorescent quantitative PCR and Western blot. Results Both restriction analysis and sequencing proved that shRNA eukaryotic expression vector pGenesil-l-Pinl （＋） was constructed correctly. The transfection efficacy of SW620 cells with pGenesil-l-Pinl （＋） was 64%. The inhibiting rate on transcription of Pin1 mRNA in SW620 cells reached a peak value （70. 2%） 72 h after transfection with plasmid pGenesil-l-Pinl（＋）, while that on expression of Pinl protein was 60%. Conclusion The shRNA interfering plasmid targeting Pinl gene was successfully constructed, which inhibited the transcription of Pinl mRNA and expression of Pinl protein in SW620 cells.