摘要：
Objective To investigate the effect of Gsh2 silencing on the sonic hedgehog (Shh) and GLI1 gene expression,which are the key factors of hedgehog pathway in human pancreatic cancer cell SW1990.Methods Three shRNAs tagerting Gsh2 (Gsh2-shRNA) were constructed and were inserted into pLKO.1 puro vector by double digestion with restriction enzyme,the empty vector without shRNA was used as a negative control.After plasmid amplification,plasmids DNA was extracted,and underwent electrophoretic separation and sequencing by double digestion.After confirmation by gene sequencing,plasmid was transfected into the human pancreatic cancer cell line SW1990,then real time PCR was used to detect the Gsh2 mRNA expression,and the plasmid with best silencing effect was selected to transfect the SW1990 cells,real time PCR and Western blot were respectively used to detect the mRNA and protein expression of the key factor in hedgehog pathway(Gsh2,Shh,Gli1).Results The gene sequencing of the 3 recombinant plasmids showed that Gsh2-shRNA were correctly constructed.If the mRNA expression of empty vector transfection cells was 1,the Gsh 2 mRNA expressions of SW1990 cells which were transfected by recombinant Gsh2-shRNA-1,Gsh2shRNA-2,Gsh2-shRNA-3 were 0.41 ± 0.02,0.22 ± 0.043,0.53 ± 0.03,and Gsh2-shRNA-2 had the best silencing effect.If the mRNA or protein expression of empty vector transfection cells was 1,the Gsh2,Shh,Gli1 mRNA expressions of cells with Gsh2-shRNA-2 transfection were 0.12 ±0.02,0.97± 0.13,0.19± 0.03,and the protein expressions were 0.55 ± 0.12,0.74 ± 0.06,0.53 ± 0.09.The Gsh2,Glil mRNA and protein expressions with Gsh2-shRNA-2 transfection were significantly down-regulated when compared with those of empty vector transfection cells,and the difference between the two groups was statistically significant (P＜0.01 or ＜0.05).Conclusions Gsh2 gene silence in pancreatic cancer cell line SW1990 can repress the expression of GLI1 in hedgehog pathway without affecting Shh expression.%目的 探讨Gsh2基因沉默对胰腺癌SW1990细胞Hedgehog (Hh)通路关键成员Shh、Gli1基因表达的影响.方法 构建3个靶向Gsh2的shRNA(Gsh2-shRNA),应用双酶切法插入质粒pLKO.1puro,以未插入shRNA的空载质粒作为阴性对照.重组质粒经扩增后,抽提质粒DNA,通过双酶切后电泳分离和测序鉴定.然后转染胰腺癌SW1990细胞,应用实时RT-PCR法检测转染细胞Gsh2 mRNA表达,筛选沉默效果最佳的插入Gsh2-shRNA重组质粒.选择最佳重组质粒转染胰腺癌SW1990细胞,采用实时RT-PCR法和蛋白质印迹法检测转染细胞的Gsh2、Shh、Gli1 mRNA和蛋白表达量.结果 3个重组质粒经双酶切电泳及测序鉴定后证实插入的Gsh2-shRNA片段正确,符合预期.以转染空载质粒细胞的mRNA表达量为1,重组质粒Gsh2-shRNA-1、Gsh2-shRNA-2、Gsh2-shRNA-3转染细胞后Gsh2mRNA表达量分别为0.41 ±0.02、0.22 ±0.043、0.53 ±0.03,以Gsh2-shRNA-2的沉默效应最佳.以转染空载质粒细胞的mRNA或蛋白表达量为1,转染Gsh2-shRNA-2细胞的Gsh2、Shh、Gli1 mRNA相对表达量分别为0.12 ±0.02、0.97 ±0.13、0.19 ±0.03;蛋白表达量为0.55±0.12、0.74 ±0.06、0.53 ±0.09.转染Gsh2-shRNA-2细胞的Gsh2、Gli1 mRNA及蛋白表达量较转染空载质粒细胞显著下调,差异均有统计学意义(P值均＜0.01),而Shh mRNA及蛋白表达量的差异无统计学意义.结论 Gsh2基因沉默可抑制胰腺癌SW1990细胞Hh通路成员Gli1的表达,而不影响Shh基因的表达.
