Detection Of The Transcriptional Activity Of Various GFAP Promoter Segments In Glioma Cells And Optimization Of A Specific Promoter For Gene Therapy

Objective:Gliomas are the most common primary central nervous system tumors,50%of which are arising from astrocytic tumors(astrocytic tuomrs,AT).Glial fibrillary acid protein(GFAP)is the major intermediate filament protein in the astrocytes,not only strongly expresses in mature astrocytes but also in glioma cells arising from astrocytes.Additionally,GFAP is the hallmark of gliomas.But in malignant gliomas,the transcriptional activities of GFAP promoter and GFAP expression are downregulated with increase of the AT malignancy degree and the dedifferentiation degree of tumor cells.In our study,transcriptional activities of different GFAP promoter segments was detected and compared in malignant AT cells(TJ905),NGF induced PC12 cells(PC12)and non-tumor cells(HEK293),than the factors influencing the efficiency were analyzed,in order to improving the biological safety of gene therapy by enhancing tissue specific expression of gene.Methods:?GFAP promoter segment-1660?+16(GFAPp)was PCR amplified using the blood of healthy persons.?For quantitativly and qualitativly detecting the transcriptional activities of GFAP promoter,pEGFP-GFAPp and pGL3-Basic-GFAPp were constructed by subcloning and inserting GFAPp into reporter gene vectors pEGFP-1 and pGL3-Basic.?With pEGFP-GFAPp as a template,the other 5 GFAP promoter segments 1549?+16(GFAPp1)?-1297?+16(GFAPp2)?-985?+16(GFAPp3)?-811?+16(GFAPp4)?-660+16(GFAPp5)were PCR amplificated.All the primers were added Hind III site(AAGCTT)at 5'end.?For quantitativly and qualitativly detecting the transcriptional activities of them,pEGFP-GFAPpl,pEGFP-GFAPp2,pEGFP-GFAPp3,pEGFP-GFAPp4,pEGFP-GFAPp5 and pGL3-Basic-GFAPp1,pGL3-Basic-GFAPp2,pGL3-Basic-GFAPp3,pGL3-Basic-GFAPp4 and pGL3-Basic-GFAPp5 were constructed by subcloning and inserting the corresponding segments into reporter gene vectors pEGFP-1 and pGL3-Basic.?Transient-transfect HEK293,TJ905 and NGF-induced PC 12 cell line with the recombination expression vectors.Fluorescent microscope and luciferase assay were used to measure the expression of these segments qualitatively and quantitatively.Results:?The 6 different GFAP promoter,-1660?+16(GFAPp),-1549?+16(GFAPp1),-1297?+16(GFAPp2),-985?+16(GFAPp3),-811?+16(GFAPp4)and-660?+16(GFAPp5),segments were successfully amplified,and had been proved by DNA sequencing.?pEGFP-GFAPp,pEGFP-GFAPpl,pEGFP-GFAPp2,pEGFP-GFAPp3,pEGFP-GFAPp4,pEGFP-GFAPp5,pGL3-Basic-GFAPp,pGL3-Basic-GFAPpl,pGL3-Basic-GFAPp2,pGL3-Basic-GFAPp3,pGL3-Basic-GFAPp4 and pGL3-Basic-GFAPp5 were successfully constructed and proved by restriction endonuclease detection.?All the 6 segments failed to derect the expression of EGFP in HEK293 cell line,while GFAPp,GFAPp4 and GFAPp5 promoted brighter fluorescent than the other 3 segments in TJ905 cell line.Quantitative analysis showed rather low transcriptional activities of all the segments in HEK293 cell line.In NGF induced PC 12 cell line,the transcriptional activities of GFAPp and GFAPpl were similar to those in HEK293 cell line(P>0.05)and significantly lower than the other 4 segments(P<0.01).All the 6 segments exhibited significantly higher promotive efficiencies in TJ905 cell line than the other cell lines examined(P<0.001),whilst the efficiencies of GFAPp4 and GFAPp5 were the highest among the six segments.Conclusions:?GFAPp,GFAPp4 and GFAPp5 segments of GFAP promoter have stronger transcriptional activities in malignant AT.?It is very important to retain B region in GFAP promoter,for keeping its effective transcription in the malignant AT cells and normal astrocytes.?-1297?-1549 zone in GFAP promoter contains the key elements regulating astrocyte-specificity of GFAP promoter in malignant AT.? C1?C3 sections inhibit the transcriptional activities of GFAP promoter in malignant AT.?-842?-638 zone in C4?C6 sections can not affect the transcription of GFAP promoter in malignant AT cells,whilst they can retain the cell-specificity of GFAP promoter.?GFAPp5,the shortest segment of GFAP promoter examined with highest transcriptional activity and specificity for cells derived from the central nervous system,could serve as an ideal promoter for malignant AT gene therapy.