靶向wnt-1基因的RNA干涉质粒构建及抑制U251细胞增殖的实验研究

Construction of RNAi recombinant expression vectors of targeting wnt-1 gene and study of its growth inhibition effect on glioma cell U251

目的构建靶向wnt-1基因的RNA干涉表达载体,转染人胶质瘤细胞U251中,研究该质粒的抑瘤效果。方法根据siRNA设计原则,通过软件设计,选取含有21个核苷酸(21nt)靶序列,形成siRNA的DNA模板并克隆到pGPU6/GFP/Neo载体中,获得靶向抑制wnt-1基因表达的siRNA表达载体,并经酶切测序鉴定。用该干涉质粒转染U251细胞,通过逆转录PCR、Western blotting方法检测wnt-1基因mRNA和蛋白表达水平,用MTT法、流式细胞术初步评价对U251细胞的增生、细胞周期影响。结果成功构建针对靶向wnt-1基因的pGPU6/GFP/Neo-shRNA-wnt-1 RNA干涉质粒,可显著抑制U251细胞wnt-1基因的mRNA和蛋白表达。MTT法分析转染细胞增生明显抑制,流式细胞仪分析细胞周期证实转染后比空白对照组受到阻滞。结论靶向wnt-1基因的pGPU6/GFP/Neo-shRNA-wnt-1 RNA干涉质粒构建成功,并可特异性的沉默wnt-1基因蛋白表达,使细胞增长减慢,细胞周期阻滞,本实验通过wnt-1的RNA干涉,为由wnt-1介导的wnt信号通路在胶质瘤发病机制中的抑瘤作用提供了依据,并为进一步该基因的基因治疗临床实验提供了可靠基础。

Objective To construct the RNAi expression vectors of wnt-1 and transfected glioma cell line U251 to study its effect by this plasmid. Methods According to the sequence of the coding region of wnt-1 gene, two strings of 21 nucleotides of inverted sequence flanking the loop sequence of two complementary 21-base oligonucleotides were designed and synthesized to form hairpin construction as the DNA templates for the target siRNA. The siRNA templates were cloned into siRNA expression vector pGPU6/GFP/NCO, and the sequence of the plasmid was identified by DNA sequencer and restriction endonuclease digestion. Thus, the resulted vector pGPU6/GFP/Neo -shRNA-wnt-1 was transfected into U251 cells. RT-PCR and Western blotting were performed to evaluate wnt-1 gene silencing induced by siRNA transfection at mRNA and the protein levels. Pre- liminary study of its growth effect on glioma cell U251 was measured by MTT and FCM. Results It was verified that the specific DNA oligonucleotide was cloned into the vector successfully, and the protein expression of wnt-1 gene in U251 cells was significantly reduced after transfecting the re- combinant plasmid, compared to the controls. Western blotting revealed significantly lowered wnt-1 expression at protein levels in transfeeted U251 cells, which exhibited a significantly higher death rate after transfection as shown by MTT . Conclusion It indicates the shRNA expression vector has been successfully established which can inhibit the protein expression of wnt-1 gene, and that provides the precondition for the further study of wnt-1 by wnt signaling pathway in glioma pathogenesis.