人PDLIM4基因启动子报告基因载体的构建及其在人类肿瘤细胞中的表达

Construction of human PDLIM4 promoter luciferase report gene vectors and their expressions in cancer cells

目的构建人PDLIM4基因启动子荧光素酶报告基因重组质粒pGL3-promoter,检测其在不同肿瘤细胞中的表达。方法据Genebank中人PDLIM4基因启动子序列分别设计上下游引物,扩增其启动子片段,并插入到pGL3-Basic报告基因载体,分别得到含有3kb和1.2kb的PDLIM4基因启动子的两个报告基因质粒pGL3-PD-LIM4-3kb和pGL3-PDLIM4-1.2kb。序列为1.2kb的启动子利用Erase-a-base试剂盒,经外切酶Ⅲ从5'端逐步酶切得到5个含不同长度启动子序列的报告基因载体:pGL3-PDLIM4-1.2D1、pGL3-PDLIM4-1.2D2、pGL3-PDLIM4-1.2D3、pGL3-PDLIM4-1.2D4、pGL3-PDLIM4-1.2D5。将构建的报告基因载体分别瞬时转染以下细胞株:Hela、MDA-MB231、KB、PC-3、LNCaP,检测其荧光素酶表达活性。结果所构建质粒经酶切、测序鉴定,其片段长度及序列均正确。转染结果显示,长片段的3kb和1.2kb启动子序列在所测试的细胞株中均有表达,但在人激素非依赖前列腺癌PC-3细胞株、人激素依赖前列腺癌LINCaP细胞株中表达较低。而不同长度的启动子报告基因质粒转染PC-3、LNCaP的结果显示,pGL3-PDLIM4-1.2kb表达活性最强,pGL3-PDLIM4-970bp表达活性最弱。结论成功构建了7个分别含有不同长度的PDLIM4启动子报告基因载体,其在不同的肿瘤细胞株中均有不同程度的表达,而在前列腺癌细胞中表达较低。

Objective To construct PDLIM4 promoter luciferase report gene vectors containing different promoter sequences,detect expressions of them in different cancer cell lines,and investigate regulatory mechanisms by which the human PDLIM4 gene is silenced in prostate cancer.Methods The PDLIM4 gene promoter was amplified from human genomic DNAs using standard PCR.Products of PCR were inserted into a luciferase report gene pGL3-Basic vector,and generated two recombinant plasmids which contained a 3 kb sequence（pGL3-PDLIM4-3 kb） or a 1.2 kb sequence（pGL3-PDLIM4-1.2 kb）.Delete mutants were generated by 5′ deletion mutagenesis according to the protocol of the ERASE-A-BASE system（Promega）.PDLIM4 promoter fragments-929/＋41,-762/＋41,-612/＋41,-604/＋41 and-586/＋41（relative to the transcription start site of PDLIM4,GenBank No.X93520） were amplified from pGL3-PDLIM4-1.2 kb.PCR products were ligated together and generated delete mutant gene report vectors,designed as pGL3-PDLIM4-1.2D1,pGL3-PDLIM4-1.2D2,pGL3-PDLIM4-1.2D3,pGL3-PDLIM4-1.2D4 and pGL3-PDLIM4-1.2D5.Promoter activities were detected after promoters were transiently transfected into different cell lines including Hela,MDA-MB231,KB,PC-3 and LNCaP.Results The constructed vectors were confirmed by restriction enzyme digestion and sequence analysis.Transient transfection results showed that promoter activities of pGL3-PDLIM4-3kb and pGL3-PDLIM4-1.2 kb were evident in cancer cell lines,and lower activity was observed in prostate cancer LNCaP and PC-3 cells when compared with the others.Also,luciferase activities of 5′-end delete mutants containing a array of sequence regions in PDLIM4 promoters were detected in prostate cancer cells.The 1.2 kb promoter displayed highest activity while the 970 bp promoter displayed lowest activity in cells compared with other tested promoter reporters.Conclusion 7 luciferase report gene vectors were successfully constructed,containing an array of fragments upstream of the PDLIM4 promoter,and displaying different promoter activities in various cell lines.Cloned 3 kb and 1.2 kb promoter regions present low activities in prostate cancer cells compared with other cell lines.