PAK1基因3′-UTR区荧光素酶报告基因载体的构建及鉴定

Construction of PAK1 3′-UTR luciferase reporter vector and identification of its activity

目的针对人p21小GTP酶活化激酶1(p21-activated kinase-1,PAK1)基因的3′端非翻译区(3′-untranslated region,3′-UTR)构建PAK1的荧光素酶报告基因载体,并对其进行鉴定和筛选,为后续PAK1与microRNA-145(miR-145)的功能和机制研究提供实验基础。方法用PCR法扩增出PAK1基因3′-UTR片段,经连接、酶切插入到pGL3载体上,构建了PAK1 3′-UTR荧光素酶报告基因载体。通过生物信息学方法预测miR-145可能靶向作用于PAK1基因的3′-UTR,后将上述重组质粒以及miRNA mimics(miRNA模拟物)、miRNA-Con(miRNA control,miRNA阴性对照)瞬转到膀胱癌T24和J82细胞系中,并检测其相对荧光素酶活性。另外,利用实时定量PCR法检测不同实验组中PAK1的表达情况。结果成功构建了重组pGL3-PAK1 3′-UTR WT质粒,并通过双酶切以及测序的方法验证所得结果的正确性。且转染pGL3-PAK1 3′-UTR WT质粒后,T24/miR-145组和J82/miR-145的相对荧光素酶活性显著低于T24/miR-con和J82/miR-con组,差异有统计学意义(P<0.05)。另外,通过实时定量PCR法发现,miR-145mimics组的PAK1表达水平显著低于miR-145con组。结论成功构建了PAK1基因3-UTR区荧光素酶报告载体,且miR-145能够显著降低其荧光素酶活性,提示miR-145能靶向负调控PAK1的表达。

Objective To construct the luciferase reporter gene vector containing 3＇-untranslated region （3＇-UTR） of p21-activated kinase-1 （PAK1）, which will provide basis for subsequent function and mechanism research on PAK1 and mi- croRNA-145. Methods The 3＇-UTR of PAK1 gene was amplified by PCR, and inserted into pGL3 control reporter,vector after link and restriction enzyme digestion. It was predicted by bioinformation method^hat miR-145 could target the 3＇-UTR of PAK1. The recombinant plasmid with microRNA mimics or microRNA control were transfected into T24 or J82 cells using Li- pofectamine 2000 to detect the relative luciferase activity. In addition, the expression of PAK1 was analysed by real-time PCR. Results The recombinant plasmid was constructed and confirmed by enzyme digestion and sequence reaction. After transfec- tion with the plasmid pGL3-PAK1 3＇-UTR WT, we found that compared with T24 or J82 cells treated with miRNA con, the luciferase activity of T24 or J82 cells transfected with miRNA mimics was significantly decreased with statistic significance. Futhermore, the expression of PAK1 in miR-145 mimics group was remarkably lower than that in miR-145 control group. Conclusions The PAK1 3r-UTR luciferase reporter vector was constructed successfully, and the luciferase activity of the re- combinant plasmid could be dramatically reduced by miR-145. It indicated that miR-145 could downregulates PAK1 expression by targeting its 3r-UTR.