摘要
目的构建galectin-1基因特异小分子干扰RNA(siRNAs)真核干扰载体,并获得干扰载体稳定转染的LST-R1细胞系。方法设计2对galectin-1 mRNA(GenBank:NM002305)特异性siRNAs,两端分别带有BamHⅠ/HindⅢ酶切位点和一个9nt的发夹结构,通过基因克隆技术将其插入真核表达质粒pSUPER-puro中,构建galectin-1 siRNA表达载体,以EcoRⅠ和HindⅢ双酶切及测序验证重组载体的正确性。将重组载体与pcDNA6/TR共转染LST-R1细胞,以嘌呤霉素(0.8μg/ml)进行阳性克隆筛选。四环素(4μg/ml)诱导48h后,RT-PCR检测galectin-1 mRNA表达水平,Western blotting和细胞免疫化学方法检测galectin-1蛋白质水平的变化。结果测序表明galectin-1 siRNA真核表达载体p-shRNA1和p-shRNA2干扰序列与读码框相符。p-shNRA1和p-shRNA2与pcDNA6/TR共转染LST-R1细胞,获得稳定的重组质粒转染细胞p-shRNA1-LST和p-shRNA2-LST。RT-PCR显示,galectin-1 mR-NA在LST-R1细胞、p-shRNA1-LST细胞、p-shRNA2-LST细胞以及p-LST细胞中的表达量分别为0.616、0.298、0.373和0.641,以p-shRNA1-LST的干扰效果最好。Western blotting结果显示,在LST-R1、p-shRNA1-LST、p-shRNA2-LST和p-LST细胞中galectin-1的表达量分别为1.00、0.07、0.38和0.97,以p-shRNA1-LST干扰效果最好,与免疫细胞化学的结果一致。结论成功构建了galectin-1真核干扰载体,RNAi载体转染LST-R1细胞系的建立为进一步研究galectin-1在大肠侧向扩散型肿瘤中的作用奠定了实验基础。
Objective To construct the RNA interference （RNAi） eukaryotic expression vectors of galectin-1, and establish the LST-R1 eell lines stably transfected by RNAi vectors. Methods Two pairs of small interfering RNAs （siRNA） targeting to galectin-1 mRNA （GenBank： NM002305） were designed. The corresponding single-strand short hairpin interfering RNAs （shRNA）, containing BamH Ⅰ, Hind Ⅲ sites and a 9nt hairpin structure, was synthesized and armealed. The annealed product and the linear eukaryotic expression plasmid pSuperior-puro, which were digested with BglⅡ and Hind Ⅲ, were ligated by T4 ligase to set up the interfering system. The recombined plasmid was identified with EcoR Ⅰand Hind Ⅲ enzyme digestion and sequencing, and co-transfected to LST-R1 cells with pcDNA6/TR with Lipofectamine2000. Positive clones were selected with 0. 8μg/ml puromycin. After incubated with 4μg/ml tetracyclin for 48 hours, RT-PCR, Western blotting and immunochemistry were employed to determine galectin-1 mRNA and protein levels. Results Sequencing results suggested that the nucleotide sequence and read frame of RNAi eukaryofic exprssion vector of galectin-1, p-shRNA1 and p-shRNA2 were perfect. Stably transfected LST-R1 cell lines of p-shRNA1-LST and p-shRNA2-LST were established. The relative values of galectin-1 expression in LST-R1 cells, p-shRNA1-LST cells, p-shRNA2-LST cells and p-LST cells by RT-PCR were 0. 616, 0. 298, 0. 373 and 0. 641, respectively, and 1.00, 0. 07, 0. 38 and 0. 97 by Western blotting. The p-shRNA1 gave the best interfering effect, which was in conformity with the results of immunochemistry measurement. Conclusions RNAi eukaryotic expression vector of galectin- 1 mRNA has been successfully constructed. Establishment of the stably transfected LST-R1 cell lines may lay a foundation to explore the roles of galectin-1 in laterally spreading tumor.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第3期307-310,共4页
Medical Journal of Chinese People's Liberation Army
