Function Study Of GHS-R Promoter In Somatotroph Adenoma

PartⅠConstructing and Identifying the GHS-R Promoter Deletion MutantsObjective: To construct a series of firefly luciferase report gene enhancer vectors for 5'flanking promoter region of GHS-R gene.Methods: A series of DNA fragments of 5'flanking promoter region of GHS-R gene were amplified from the genomic DNA of primary cultured human pituitary somatotrophinomas cells in PCR. The PCR products were then cloned into the pGL3-Enhancer vector.Results: Restriction enzymes digestion and nucleotide sequencing confirmed that the recombinant plasmids pGL3-Enhancer A~F had been constructed.Conclusion: A new methods was provided to study the transcription regulation of GHS-R gene in vitro. A foundation for further study of GHS-R gene promoter in human pituitary somatotrophinomas was established. PartⅡPrimary Function Analysis of GHS-R PromoterObjective: To analyze the structure and function of GHS-R promoter region primarily, using the constructed recombinant reporter gene promoter-pGl3-enhancer vectors containing different length of GHS-R promoter deletion mutants.Methods:①The recombinant plasmid pGL3-Enhancer-A containg the shortest length of human GHS-R promoter deletion mutants and pGL3-Enhancer-F containg the longest length of human GHS-R promoter deletion mutants were transfected into 293 cell, 95D cell and GH3 cell with the liposomes.②The promoter activities of different recombinant plasmids were detected by the Dual Luciferase Reporter Assay System, so the cell specificity of GHS-R promoter was identified.③The recombinant plasmids containg different length of human GHS-R promoter deletion mutants were transfected into GH3 cell.④The promoter activities of different recombinant plasmids were detected by the Dual Luciferase Reporter Assay System, then the important transcriptional regulation region of GHS-R promoter were identified.Results:①After transfected pGL3-Enhancer-A plasmid, we found that there was not significant difference between the promoter activities of various cell in statistics(p>0.05). After transfected pGL3-Enhancer-F plasmid, we found that the promoter activity of GH3 cell increased markedly compared with 293 cell and 95D cell.②According to the Dual Luciferase Reporter Assay System, we found that the promoter activities increased markedly between pGL3-Enhancer-B(8.4±0.86)and pGL3-Enhancer-A(2.3±0.21),p<0.01; pGL3-Enhancer-D(10.6±0.54) and pGL3-Enhancer-C(5.2±0.30), p<0.05; pGL3-Enhancer-E(14.1±0.74)and pGL3-Enhancer-D(10.6±0.54), p<0.05, and also observed that the promoter activities declined significantly between pGL3-Enhancer-C(5.2±0.30)and pGL3-Enhancer-B(8.4±0.86), p<0.05, the promoter activities declined between pGL3-Enhancer-F(12.9±0.23)and pGL3-Enhancer-E(14.1±0.74), but there was not significant difference, p>0.05.Conclusion: Human GHS-R promoter(-1008~+10)has specific expression activity in GH3 cell; There were not regulation elements between -168~+10 in the upstream of the GHS-R promoter; there was a positive transcriptional regulation region from -254 to -168, -625 to -355, -910 to -625 respectively, and a negative transcriptional regulation region between -355 to -254 in the upstream of the human GHS-R promoter. PartⅢExperimental study of hormone related cis-element in the GHS-R promoter regionObjective: To investigated the hormone regulation of GHS-R promoter, and identify the specific sequences of hormone related cis-element in the GHS-R promoter region.Methods:①Using various agents interfered the process that pGL3-Enhancer-F plasmid was transiently transfected GH3 cells.②We analyzed the effects of various agents by detecting the activity of promoter.③Plasmids containing progressively decreasing amount of GHS-R promoter region upstream of the luciferase gene were transiently transfected into GH3 cells, with or without hormonal treatment.④The promoter activities of different recombinant plasmids were detected by the Dual Luciferase Reporter Assay System, so the specific sequences of the hormone related cis-elements in the GHS-R promoter region were identified.Results:①Treatment with hydrocortisone significantly inhibited the promoter activities of pGL3-Enhancer-F; treatment with T3 orβ-estradiol significantly enhanced the pGL3-Enhancer-F; and treatment with forskolin, somatostatin, TPA and IGF-1 did not significantly influence activity of the GHS-R promoter region analyzed.②With or without hydrocortisone , we observed that the ratio of relative activity declined significantly between pGL3-Enhancer-C(0.7±0.10)and pGL3-Enhancer-B(1.2±0.17); with or without T3, we found that ratio of relative activity increased markedly between pGL3-Enhancer-B(1.8±0.22)and pGL3-Enhancer-A(1.2±0.16); with or withoutβ-estradiol, we observed that ratio of relative activity increased markedly between pGL3-Enhancer-B(2.0±0.17)and pGL3-Enhancer-A(1.2±0.11).Conclusion: A negative glucocorticoid-responsive element may be located in the GHS-R promoter region between -355 and -254; Positive thyroid and estrogen responsive elements may be located in the GHS-R promoter region between -254 and -168.