Abstract:

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 3kb sequence (pGL3-PDLIM4-3kb) or a 1.2kb sequence (pGL3-PDLIM4-1.2kb). 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PDLIM4-1.2kb. 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-PDLIM4-1.2kb 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 1.2kb promoter displayed highest activity while the 970bp 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 3kb and 1.2kb promoter regions present low activities in prostate cancer cells compared with other cell lines.

Key words: Gene, PDLIM4; Promoter; Report gene vector; Luciferase