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背景:有研究表明富含亮氨酸重复序列免疫球蛋白样蛋白1(leucine-rich repeats and immunoglobulin-like domains1,LRIG1)基因在神经胶质瘤细胞中呈低表达,LRIG1基因过表达后对神经胶质瘤细胞的LRIG1mRNA和蛋白表达均明显增强和抑制其生物学行为,但却很少有研究从阻断LRIG1基因的表达的角度进行论证。目的:构建针对LRIG1基因的特异性RNA干扰质粒,稳定转染人脑胶质瘤GL15细胞系,观察其对目的基因LRIG1表达的影响。方法:根据GenBank提供的LRIG1基因序列设计2条RNA干扰序列,命名为LRIG1-shRNA1和LRIG1-shRNA2,并设计1条非特异性序列作为阴性对照,命名为pGenesil2-negativeshRNA。合成各自的寡核苷酸链,退火后与pGenesil2质粒载体连接,转化扩增后测定序列。用不同浓度的G418作用于GL15细胞确定G418对GL15细胞的筛选浓度。将3种重组表达载体转染GL15细胞,G418筛选后挑单克隆并扩增获得稳定株。Western blot检测LRIG1蛋白的表达。结果与结论:构建的重组pGenesil2-LRIG1-shRNA质粒经限制性酶切及DNA测序分析证明其序列插入正确。转染pGenesil2-LRIG1-shRNA1(LRIG11)细胞和pGenesil2-LRIG1-shRNA2(LRIG12)细胞LRIG1蛋白表达水平较阴性对照组pGenesil2-negativeshRNA分别下降47.9%(P<0.01)和32.8%(P>0.05)。结果证实,实验成功构建了针对LRIG1基因的特异性shRNA表达载体pGenesil2-LRIG1-shRNA1,其转染GL15细胞后可抑制LRIG1的表达。
BACKGROUND: Studies have shown that the gene encoding leucine-rich repeats and immunoglobulin-like domains1 (LRIG1) is low expressed in glioma cells. After LRIG1 gene overexpression, LRIG1 mRNA and protein expression of glioma cells were significantly enhanced and inhibited its biological behavior, but few studies from the perspective of blocking the expression of LRIG1 gene to demonstrate. OBJECTIVE: To construct a specific RNAi plasmid targeting LRIG1 gene and stably transfected it into human glioma GL15 cell line to observe its effect on the expression of target gene LRIG1. Methods: According to the sequence of LRIG1 gene provided by GenBank, two RNA interference sequences were designed and named as LRIG1-shRNA1 and LRIG1-shRNA2. One nonspecific sequence was designed as a negative control and named as pGenesil2-negativeshRNA. The respective oligonucleotide strands were synthesized, annealed and ligated with pGenesil2 plasmid vector, and the sequences were determined after transformation and amplification. The concentration of G418 on GL15 cells was determined using different concentrations of G418 on GL15 cells. Three kinds of recombinant expression vectors were transfected into GL15 cells, single clones were screened by G418 and amplified to obtain stable strains. Western blot detection of LRIG1 protein expression. RESULTS AND CONCLUSION: The constructed recombinant pGenesil2-LRIG1-shRNA plasmid was verified by restriction enzyme digestion and DNA sequencing. LRIG1 protein expression in transfected pGenesil2-LRIG1-shRNA1 (LRIG11) cells and pGenesil2-LRIG1-shRNA2 (LRIG12) cells decreased by 47.9% (P <0.01) and 32.8% (P> 0.05) compared with the negative control group. The results confirmed that a specific shRNA expression vector pGenesil2-LRIG1-shRNA1 targeting LRIG1 gene was successfully constructed and successfully transfected into GL15 cells to inhibit LRIG1 expression.