| Stathmin is overexpressed in nearly all of human malignancies including osteosarcoma, uterine cervix cancer, breast cancer, lung cancer. The overexpression of stathmin gene has led to many changes of malignant phenotype in many malignancies. Our previous studies have demonstrated that inhibition of stathmin expression in cancer cells can abolish their transformed phenotype and arrest cell proliferation, however, the mechanisms of stathmin gene overexpression in a variety of human malignancies are not fully clear. Therefore, in this study, we investigated the mechanisms of transcriptional regulation of stathmin gene overexpression.Objective The computer-assisted analysis of the core promoter of stathmin gene identified four potential binding sites for E2F1, NF-Y, Sp1 and Egr1. To investigate the mechanisms of transcriptional regulation of stathmin gene overexpression, we explored the regulation of stathmin gene promoter by E2F1, NF-Y, Sp1 and Egr1, which may have a great significance for the function research of stathmin gene and set up a new approach for cancer therapy. Methods (1) Stathmin gene promoter fragment was amplified by PCR method using A549 cells genomic DNA as template. The recombinant vectors with stathmin gene promoter (pGL3-Basic/LUC/stap and pGL3-Basic/EGFP/stap) were constructed and transfected into 293 cells and A549 cells. Then the activity of stathmin gene promoter was detected using reporter (luciferase and green fluorescence protein) assay. (2) Five fragments with different lengths of stathmin gene promoter were amplified by PCR method, and then subcloned into pGL3-Basic/LUC and pGL3-Basic/EGFP vector respectively by recombinant DNA technique. The activity of reporter gene of these recombinant vectors bearing truncated stathmin gene promoters was detected using reporter (luciferase and green fluorescence protein) assay to determine the sequence of stathmin gene core promoter. According to these results, the sequence of core promoter will be determined. (3) The computer-assisted analysis of the core promoter of stathmin gene identified four potential binding sites for E2F1, NF-Y, Sp1 and Egr1. Site-specific mutations were carried out at the binding sites for four transcription factors in stathmin gene core promoter respectively, and then these mutant fragments were introduced into pGL3-Basic/LUC and pGL3-Basic/EGFP vectors respectively. The activity of reporter gene of these recombinant vectors was detected using reporter assay, and the direct binding evidence of E2F1, NF-Y, Sp1 and Egr1 in stathmin gene promoter was confirmed by Chromatin immunoprecipitation (ChIP). (4) To construct the expression vectors of full length wild type E2F1, NF-Y, Sp1 and Egr1, cDNAs of these transcription factors were amplified by PCR method. The PCR products were then inserted into the EcoRI and XhoI sites of pcDNA3.1(+) vector. DNA templates encoding shRNA of these transcription factors were designed and synthesized, and shRNAs were subcloned into BamHI and HindIII sites of pSilencer4.1-CMVneo vector. All inserted sequences were verified by DNA sequencing. These recombinant vectors were transfected into A549 cells and transfected cells were selected in G418 for 14 d. The positive clones were obtained after G418 selection. The level of stathmin gene expression in the positive clones was detected by RT-PCR and Western blot methods. (5) To explore the relationship between stathmin and Egr1, the changes of stathmin gene mRNA and protein levels were detected in response to Egr1 activation by DOX, PMA and UV. Site-specific mutations were also performed for Egr1 binding sites in stathmin gene promoter, and introduced into pGL3-Basic/LUC/stap. The activity of reporter gene of the recombinant vector was detected using reporter assay. To further verify that stathmin gene is an Egr1 target gene, ChIP and EMSA assays were performed to confirm whether Egr1can directly bind to Egr1 binding site in stathmin gene promoter. To investigate the functional role of p53-regulated pathway in stathmin gene expression, A549-KE (knock down Egr1) cells and A549 (wild type Egr1) cells were used to test the p53 regulation effect on the stathmin gene promoter activity. The expression vectors (wild type p53 or control vector) driven by CMV promoter were cotransfected with pGL3-Basic/LUC/stap vector. The changes of stathmin gene protein levels were detected in response to p53 upregulation by DOX, UV and the expression vector with wild type p53.Results (1) Stathmin gene promoter fragment was obtained by PCR and sequences were verified by DNA sequencing. The recombinant vectors with stathmin gene promoter (pGL3-Basic/LUC/stap and pGL3-Basic/EGFP/stap) were successfully constructed and transfected into 293 cells and A549 cells. The results showed that the activity of stathmin gene promoter was higher than survivin gene promoter using reporter (luciferase and green fluorescence protein) assay. (2) Five fragments with different lengths (1500 bp, 1000 bp, 600 bp, 400 bp and 200 bp) of stathmin gene promoter were obstained by PCR, and then were successfully subcloned into pGL3-Basic/LUC and pGL3-Basic/EGFP vector. According to these results of reporter assay, the activity of a 400 bp stathmin gene promoter fragment was higher than all other fragments. (3) Site-specific mutations were successfully performed for the binding sites of E2F1, NF-Y, Sp1 and Egr1 in stathmin gene core promoter, and were also successfully cloned into pGL3-Basic/LUC and pGL3-Basic/EGFP vectors. These results of reporter assay show that the activity of reporter gene was downregulated after the mutation of E2F1, NF-Y and Sp1 binding sites, but the activity of reporter gene was upregulated after the mutation of Egr1 binding sites. Chromatin immunoprecipitation (ChIP) assay showed that E2F1, NF-Y, Sp1 and Egr1 can directly bind to stathmin gene promoter. (4) The expression vectors of wild type E2F1, NF-Y, Sp1 and Egr1 as well as siRNA eukaryotic expression vectors respectively targeting E2F1, NF-Y, Sp1 and Egr1, were successfully constructed. These recombinant vectors were separately transfected into A549 cells and selected in G418. RT-PCR and Western Blot results showed that E2F1, NF-Y and Sp1 can increase the level of stathmin gene expression, and Egr1 can decrease the level of stathmin gene expression. (5) Increase of endogenous and exogenous Egr1 resulted in decreased expression of stathmin gene on both mRNA and protein level in A549 cells. But reduction of endogenous Egr1 markedly attenuated PMA-induced decrease of stathmin gene expression. Egr1 can regulate the activity of stathmin gene promoter. ChIP and EMSA results also showed Egr1 directly bound to Egr1 binding sites in stathmin gene promoter. The activity of stathmin gene promoter was decreased by wild type p53 in A549 cells, while the control vector did not change the activity of stathmin gene promoter. DOX and UV also downregulated the expression of luciferase in A549 cells, while DOX and UV did not change the activity of luciferase in A549-KE (knockdown Egr1) cells. The activity of stathmin gene promoter was markedly decreased by endogenous p53. Induction of Egr1 following p53 upregulation significantly decreased protein level of stathmin in A549 cells. In contrast, p53 upregulation did not change protein level of Egr1 and stathmin in A549-KE cells. DOX and UV also got the same result.Conclusion (1) Stathmin gene promoter fragment was obtained, and the activity of stathmin gene promoter was determined using reporter assay. The activity of stathmin gene promoter was higher than survivin gene promoter. (2) The sequence of core promoter was determined by reporter assay. (3) E2F1, NF-Y, Sp1 and Egr1can directly bind to stathmin gene promoter, and they can also regulate the activity of stathmin gene promoter. (4) E2F1, NF-Y and Sp1 can increase the level of stathmin gene expression, and Egr1 can markedly decrease the level of stathmin gene expression. (5) Transcription factor Egr1 can regulate stathmin expression, and p53 mediates the transcriptional repression of stathmin gene by Egr1. Taken together, we explored the regulation of stathmin gene in this study, which may have a great significance for the function research of stathmin gene and set up a new approach for cancer therapy. |
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